Lactic bacteria producing exopolysaccharides

ABSTRACT

DNA fragment of genomic origin coding for at least one enzyme involved in the biosynthesis of an EPS, and capable, following the transformation of a lactic bacteria, of restoring the production of an EPS in the said bacterium not initially producing any EPS, or of modifying the structure of the EPS initially produced by the said bacterium. Proteins of the Streptococcus thermophilus strain CNCM I-1590 encoded by the chromosome and which are involved in the biosynthesis of the EPS having the composition Glc:Gal:GalNac=1:2:1. Method for the manufacture of a new EPS, in which a DNA fragment coding partially or totally for at least one enzyme involved in the biosynthesis of an EPS is cloned into a vector, lactic bacteria producing another EPS are transformed with the recombinant vector, and a lactic bacterium producing a new EPS is then selected.

This is a division of application Ser. No. 08/597,236, filed Feb. 6,1996.

TECHNICAL FIELD

The present invention relates to the use of chromosomal DNA fragments oflactic bacteria coding for at least one enzyme involved in thebiosynthesis of exopolysaccharides, as well as to enzymes encoded bythese fragments.

Prior art

Lactic bacteria are known to be capable of producing two classes ofpolysaccharides in their culture medium, namely homopolysaccharides suchas dextrans or levans which consist of the repeated assembly of a singlesugar, and heteropolysaccharides commonly called exopolysaccharides orEPSs (EPS is short for the term "exopolysaccharide") consisting of theassembly of several different sugars forming a repeating unit (CerningJ., Bacteeries lactiques, Lactic bacteria!, Vol I, by Rossart H andLuquet F. M., Lorica, 309-329, 1994).

A lactic bacterium producing an EPS can impart a ropy character and/or asmooth and creamy texture to an acidified milk (Cerning et al., FEMSMicrobiol., 87, 113-130, 19/90). EPSs can also display biologicalactivities which are especially advantageous for human or animal health,such as antitumour or probiotic activities, for example (Oda M. et al.,Agric. Biol. Chem., 47, 1623-1625, 1983; EP94870139.6).

Moreover, the industry is confronted by a genetic instability of thebiosynthesis of EPSs in lactic bacteria. This generally manifests itselfduring a fermentation by the loss of EPS production by all or part ofthe lactic bacteria (see "Cerning J." above). Industrial fermentedproducts are thus subject to variations in their EPS content, which isnot always acceptable. To remedy these problems, the industry resorts atthe present time to the isolation and periodic characterization of itsbacteria so as to separate the ones which have lost their originalcharacter.

EPS biosynthesis in mesophilic lactic bacteria, that is to say lacticbacteria having optimal growth at 28°-37° C., involves at least oneenzyme which effects the linking of the sugars. No chromosomal orplasmid gene of mesophilic lactic bacteria coding for such an enzyme hasyet been identified and sequenced, although plasmids involved in EPSbiosynthesis are known.

Thus, WO 92/02142 discloses the existence of the plasmid pHV67 whichproduces in Lactococcus lactis subsp. lactis (mesophile) a substancecapable of increasing the viscosity of a fermented milk. U.S. Pat. No.5,066,588 describes two plasmids originating from a strain ofStreptococcus cremoris (mesophile) capable of imparting a thickeningcharacter on a Streptococcus lactis. Similarly, Vescovo et al. havedemonstrated a plasmid from a Lactobacillus casei subsp. casei strain(mesophile) coding for a Muc+ phenotype, that is to say for functionslinked to the production of exocellular thickeners (Vescovo et al.,Biotechnology Letters, Vol II, 709-712, 1989).

Lastly, Van den Berg et al., are seeking to isolate from a Lactobacillussake (mesophile) a group of chromosomal genes involved in thebiosynthesis of an EPS (Van den Berg D. J. C. et al., FirstInternational Conference on Polysaccharide Engineering, Trondheim,Norway, Jun. 6-8, 1994). However, no gene has yet been identified and/orsequenced.

Furthermore, EPS biosynthesis in thermophilic lactic bacteria, that isto say lactic bacteria having optimal growth at 37°-45° C., is not yetwell known. It is known, however, not to be associated with a plasmid.Thus, Vescovo et al. showed that the Muc+ phenotype of Lactobacillusdelbrueckii subsp. Bulgaricus strain 2o1 (thermophile) is linked tochromosomal functions (Vescoso et al., Biotechnology Letters, Vol II,709-712, 1989).

Thus, to date, no chromosomal or plasmid gene or group of genes codingfor an EPS of mesophilic or thermophilic lactic bacteria has beenidentified and/or sequenced.

Hence it would be very advantageous to have means for restoring orstabilizing the original EPS production in lactic bacteria. Furthermore,it would also be advantageous to have means for modifying the structureof an EPS, and thereby creating new EPSs capable of having advantageousproperties.

SUMMARY OF THE INVENTION

The objective of the invention is to provide new means for controlling,modifying and/or restoring EPS synthesis in vivo and in vitro.

To this end, the present invention relates to any lactic bacterial DNAof chromosomal origin coding for at least one enzyme involved in thebiosynthesis of the EPS possessing the repeat structure ##STR1## wheren>1; A is chosen from the group composed of β-D-Galp, β-D-Glcp and theiracetyl and phosphatyl derivatives; and x and y=2, 3, 4, 5 or 6, giventhat x≠y.

Another subject of the present invention relates to recombinant vectorscomprising a DNA fragment according to the present invention.

Another subject of the present invention relates to a protein capable ofbeing involved in the biosynthesis of the EPS having the repeatstructure ##STR2## the said protein having the amino acid sequencechosen from the group composed of the sequences SEQ ID NO: 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 and the homologous sequences (sequencespresented in the sequence listing below).

Another subject of the present invention relates to a lactic bacteriumcomprising, integrated in its chromosome or with the aid of a replicableplasmid, a DNA fragment according to the invention.

Another subject of the present invention relates to a method for theproduction of an EPS, in which (1) a DNA fragment coding for the enzymesaccording to the invention is cloned into a vector, the said vectorcomprising, in addition, a sequence permitting autonomous replication ina host cell or integration into the latter, (2) a host cell istransformed with the said vector, and (3) the transformed host cell isthen cultured under suitable conditions for the production of an EPS.

The invention also relates to another method for the production of a newEPS, in which (1) a DNA fragment coding for at least one enzyme involvedin the biosynthesis of an EPS is cloned into a vector, (2) a lacticbacterium is transformed with the said vector, and (3) the transformedlactic bacterium is then cultured under suitable conditions for theproduction of a new EPS.

Hence the present invention opens up the possibility of using DNAfragments according to the invention to restore or modify EPS productionin a lactic bacterium. Thus it is possible to envisage expressing oroverexpressing the DNAs according to the invention in a lacticbacterium, to produce EPSs intended for thickening, and making creamy,drinks or food such as liquid desserts, yoghurts, soups, dairyicecreams, coffee creams, sauces or mayonnaises, for example.

The present invention also makes it possible to have new means foridentifying chromosomal genes of lactic bacteria involved in EPSbiosynthesis.

Lastly, the present invention also provides new enzymes involved in thebiosynthesis of the EPS which is described above. These enzymes may thusbe advantageously used to synthesize or modify in vitro a polysaccharidesuch as an oligosaccharide or an EPS, for example (Ichikawa Y. et al.,American Chemical Society, 114, 9283-9289, 1992).

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1.A. is a physical map of the operon involved in the synthesis ofthe EPS of the S. thermophilus strain CNCM I-1590. The promoters andterminators are represented, respectively, by flags and hairpins. Thevertical arrow indicates the position of the insertion site of thetransposon Tn916. The horizontal arrows indicate the presence ofpotential open reading frames (ORFs). The names of the genescorresponding to the ORFs appear below the arrows. The restrictionenzymes are shown in abbreviated form (S=SacI; H=HindIII; E=EcoRI;B=BamHI).

FIG. 1.B. is a representation of the chromosomal inserts of the strainCNCM I-1590, present in the 11 pFS vectors. P1, P2 and P3 indicate theposition of the probes which are used during screening.

FIG. 1.C. is a representation of the genomic insert pFS101 comprisingthe whole of the eps operon from the SacI restriction site to BamHI,which is cloned into pJIM2279.

FIG. 2. is a representation of the optical density at 485 nm of the gelfiltration chromatography fractions comprising the sugars produced byLactococcus lactis strain MG1363 transformed with pFS101 or pJIM2279.Fraction 9: 2×10⁶ daltons (Da); fractions 11-13: 5×10⁵ Da; fractions14-16: 7.2×10⁴ Da; fractions 17-18: 4×10⁴ Da; fraction 19 and above:<5×10³ Da.

DETAILED DESCRIPTION OF THE INVENTION

In the description which follows, the term "EPS" denotes anexopolysaccharide produced by a lactic bacterium which consists of theassembly of several different sugars forming a repeating unit.

The terms acetyl and phosphatyl derivatives are used to denote galactoseor glucose comprising at least one acetyl and phosphatyl radical atpositions C₂ to C₆ on the sugar ring.

For the purposes of the present invention, "homologous sequence" isunderstood to mean any nucleic acid or amino acid sequence having anidentical function, differing from the sequences according to theinvention only in the substitution, deletion or addition of a smallnumber of nucleic acid or amino acid bases, for example 1 to 500 basepairs (bp) or 1 to 150 amino acids.

In this context, two DNA sequences which, as a result of the degeneracyof the genetic code, code for the same polypeptide will be considered,in particular, to be homologous. Similarly, two functional proteinswhich are recognized by the same antibody, the ratio of the values forintensity of recognition of the two proteins by the antibody notexceeding 1000, and preferably 100, for example, will be considered tobe homologous.

A sequence will also be considered to be homologous if it displays morethan 70% homology with the sequences according to the invention,especially more than 80% or 90%. In the latter case, the homology isdetermined by the ratio of the number of bases or of amino acids of ahomologous sequence which are identical to those of a sequence accordingto the invention, to the total number of bases or of amino acids of thesaid sequence according to the invention.

For the purposes of the present invention, "fragment which hybridizes"is understood to mean any fragment capable of hybridizing with thefragments according to the invention by the Southern blotting method(Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, U.S.A., 1989., chapters 9.31 to 9.58).Preferably, the hybridization is conducted under stringent conditions soas to avoid nonspecific or unstable hybridizations.

Lastly, the term "fragment" or "DNA fragment" should be understood to bea double-stranded DNA of chromosomal origin, which may be synthesized,reproduced in vitro, for example, by the known method called the"polymerase chain reaction", or reproduced in vivo in a bacterium of theEscherchia coli, Lactococcus lactis or Streptococcus thermophilus type,for example.

To select a DNA fragment according to the present invention, it ispossible to build a library of large DNA fragments from a lacticbacterium producing an EPS in a lactic bacterium not producing any EPS,and then to select the clone or clones producing an EPS. To this end,the genomic DNA of a lactic bacterium producing an EPS is digested witha restriction enzyme which is specific for a relatively rare restrictionsite (BamHI, SalI, PstI) or by a partial digestion with Sau3A, forexample. The digestion product is cloned into an expression orintegration plasmid which accepts large fragments (plasmid pSA3described in Example II), the recombinant plasmids are introduced intothe same species of lactic bacterium not producing any EPS, at least onetransformed clone producing an EPS is selected and the DNA fragmentresponsible for EPS production is then identified, isolated andsequenced in a traditional manner.

In view of the fact that the DNA fragments according to the presentinvention are capable of being large-sized, since they can contain agroup of genes involved in EPS biosynthesis, it may be preferable tointroduce the recombinant plasmids into the same strain of lacticbacterium from which the fragments originate, apart from the fact thatthis strain has lost the capacity to produce EPSs following a mutagenictreatment (UV or chemical treatment or treatment with a transposon).

An alternative to the method described above can also consist inbuilding a plasmid library of DNA fragments from a strain of lacticbacterium producing an EPS, in transforming the same strain of lacticbacterium with the plasmids incapable of replicating therein, inselecting the transformants which have integrated a plasmid into theirgenome by homologous recombination (selection by a resistance to anantibiotic, for example), in selecting the transformants no longerproducing any EPS and then in isolating and sequencing the chromosomalDNA fragments of the selected transformants which are adjacent to theintegrated plasmid. To this end, it is possible to digest the chromosomeof the transformants, to ligate it and then to perform a reverse PCRusing probes specific for the integrated plasmid or to introduce theligation product into a strain in which the recircularized plasmid iscapable of replicating, for example.

Another alternative to the selection method described above can alsoconsist in transforming lactic bacteria producing an EPS with a plasmidcomprising a transposon, in subjecting the bacteria to conditions underwhich the transposon is excised from the vector and integrates at randominto the genome, in selecting the clones of bacteria which have lost thecapacity to produce EPSs, and in isolating the genomic DNA fragmentsfrom the said clones into which a transposon has integrated. This methodis described in greater detail in Example I presented below.

It should be pointed out that the selection methods described brieflyabove may be applied to all known lactic bacteria, in particular tomesophilic lactic bacteria such as, for example, Streptococcus cremoris,Streptococcus lactis, Lactobacillus casei subsp. casei and Lactobacillussake, and thermophilic lactic bacteria such as, for example,Streptococcus thermophilus, Lactobacillus delbruecki subsp. bulgaricusand Lactobacillus helveticus. To this end, a person skilled in the arthas transformation techniques at his disposal for each species of lacticbacterium, and especially for Lactobacillus delbruecki subsp. bulgaricus(Sasaki Y. et al., FEMS Microbiology Reviews, 12, Fourth Symposium onLactic Acid Bacteria, Noodwijkerhout, The Netherlands, Sept. 1993).

Furthermore, the selection methods described above make it possible,more often than not, to isolate only a portion of a gene or of a groupof genes involved in the biosynthesis of an EPS. Nevertheless, a personskilled in the art may readily identify the remaining portion of thegene or group of genes by selecting in a chromosomal library, usingnucleic acid probes based on an isolated fragment, one or more clonescontaining the remaining portion, for example (see Example I.6).

It was thus possible to characterize a DNA sequence of 15.2 kb of theStreptococcus thermophilus strain deposited on 7th Jun. 1995 with theCollection Nationale de Culture de Microorganisme (C.N.C.M.) NationalCollection of Microorganism Cultures! (CNCM), Pasteur Institute, 28 ruedu Dr Roux, 75724 Paris cedex 15, France, where it received the depositNo. CNCM I-1590. Moreover, this Gram-positive strain in displays underthe microscope an appearance of non-flagellated cocci forming smallchains. This strain does not make spores and is a facultative anaerobe.

This sequence of 15.2 kb comprises genes coding for new enzymes involvedin the biosynthesis of an EPS having the repeat structure ##STR3##

Nucleotides 648 to 15250 of this sequence of 15.2 kb are shown in thesequence SEQ ID NO: 1 given in the sequence listing below. 13 completegenes are delimited in the nucleic acid sequence SEQ ID NO:1 bynucleotides 352-1803, 1807-2535, 2547-3239, 3249-3995, 4051-4731,4898-5854, 6425-7540, 7736-8212, 8221-9192, 9285-10364, 10392-11339,11302-12222 and 12233-13651.

It was possible to show that all or part of the sequence SEQ ID NO: 1makes it possible, following a transformation, to restore an EPSbiosynthesis in a host cell such as a mesophilic or thermophilic lacticbacterium which was initially not producing any EPS, in particular in aStreptococcus or a Lactococcus. As an example, the DNA sequenceaccording to the invention may thus be used to restore EPS production ina mutant of the S. thermophilus strain CNCM I-1590 no longer producingany EPS (natural mutant or one originating from a mutagenesis.

To restore the biosynthesis of an EPS, all or part of the sequence SEQID NO:1 comprising at least one of the abovementioned genes may beintegrated into a host cell by means of the method described in EP564,966, the said method being incorporated by reference in the teachingof the present invention. Briefly, this method makes it possible to beable (1) to transform the host cell with a donor plasmid which does notreplicate therein, the said plasmid comprising the said fragmentfunctionally integrated (the reading frame is preserved) into a portionof an operon originating from the host cell; (2) to identify thetransformants comprising the whole of the plasmid, integrated; (3) toselect transformants comprising only, integrated into the chromosome,the fragment according to the invention, the other sequences of theplasmid having been excised from the chromosome; and (4) to culture theselected transformants under suitable conditions for the production ofan EPS.

It may be noted that this method makes it possible not to use functionalpromoter and translation activator sequences. Furthermore, the cultureconditions suitable for EPS production are within the capacity of aperson skilled in the art, who can use standard culture media and choosethe pH, temperature and agitation of the optimum medium according to thestrain used.

It is also possible to choose to clone all or part of the sequence SEQID NO:1 comprising at least one of the abovementioned genes into aself-replicating expression plasmid downstream of functional promoterand translation activator sequences and, where appropriate, upstream ofa terminator, and then to transform a host cell with the recombinantplasmid.

Moreover, it may be observed that the EPS produced by a host celltransformed with the sequence SEQ ID NO:1, for example a Lactococcuslactis not initially producing an EPS, may be different from the EPSwhich should normally be synthesized by the recombinant enzymes, in thisinstance the EPS produced by the strain CNCM 1-1590. The use of all orpart of the sequence of 15.2 kb can hence permit the creation ofvariants of the EPS described above.

Similarly, it could be shown that all or part of the sequence SEQ IDNO:1 can also make it possible, following a transformation, to modifythe repeat structure of an EPS initially produced by a host cell, forexample by a mesophilic or thermophilic lactic bacterium, in particulara Streptococcus or a Lactococcus.

These observations thus open up the possibility of producing a novelmethod of production of a new EPS, in which (1) a DNA fragment codingpartially or totally for at least one enzyme involved in thebiosynthesis of an EPS is cloned into a vector; (2) lactic bacteria aretransformed with the recombinant vector; (3) where appropriate, a lacticbacterium producing a new EPS is selected; and (4) the transformedlactic bacterium is then cultured under suitable conditions for theproduction of a new EPS. Preferably, the vector codes for the proteinsaccording to the invention. Furthermore, the lactic bacterium canproduce an EPS other than the one synthesized by the proteins encoded bythe said vector.

In particular, a DNA fragment coding partially for at least one enzymeinvolved in the biosynthesis of a first EPS is cloned into anintegration vector, the recombinant vector is introduced into mesophilicor thermophilic lactic bacteria capable, where appropriate, of producinga second EPS via one or more chromosomal or plasmid genes, the bacteriawhich have integrated the integration vector into their chromosome areisolated, and those which produce a new EPS are then selected on accountof the inactivation of one or more genes involved in the biosynthesis ofthe second EPS. Preferably, the first and the second EPS are identical,and a DNA fragment coding partially (at least 15 base pairs) for atleast one enzyme involved in the addition of a sugar to the side chainof the repeating unit or in the modification of a sugar, such as asulpho-, phospho- or acetyltransferase, for example, is chosen.

Similarly, a DNA fragment coding totally for at least one enzymeinvolved in the biosynthesis of a first EPS may be cloned into areplicative expression vector, the recombinant vector may be introducedinto mesophilic or thermophilic lactic bacteria capable, whereappropriate, of producing a second EPS via one or more chromosomal orplasmid genes, the bacteria containing the replicative vector may beisolated, and those which produce a new EPS may then be selected onaccount of the expression of one or more genes involved in thebiosynthesis of the first EPS. Preferably, DNA fragments coding forenzymes involved in the modification of a sugar, such as a sulpho-,phospho- or acetyltransferase, for example, or in the addition of therepeating unit of a sugar such as a glucosyl- or agalactosyltransferase, for example, are chosen.

Preferably, at least one of genes carried by the sequence SEQ ID NO:1 isused totally or partially. At least one plasmid gene of mesophiliclactic bacteria involved in the biosynthesis of an EPS (gene which maybe sequenced from known plasmids) may also be used.

Lastly, the recombinant vector can be any linear or circular, single- ordouble-stranded expression or integration DNA fragment comprising a DNAsequence according to the invention, in particular all or part of thesequence SEQ ID NO: 1. In the event of the method described in EP564,966 not being used, care should be taken that the vector can expressthe DNA according to the invention through appropriate nucleic acidsequences (promoter; ribosome binding site; preferred codon) and, whereappropriate, that it comprises one or more origins of replication fromvarious bacteria, in particular from Escherichia coli and/or from aStreptococcus, for example.

The invention also relates to new enzymes encoded by the genes of thesequence SEQ ID NO:1, in particular the sequences which are homologouswith them. Their use to modify or synthesize in vitro an oligosaccharideor a polysaccharide such as an EPS, for example, may thus be envisaged.For this purpose, it is preferable to purify at least one of theseenzymes, by overexpressing their gene in a traditional manner in abacterium and isolating them in a traditional manner, by precipitationand/or chromatography of the cultural medium, for example.

Another subject of the present invention relates to a lactic bacteriumcomprising, integrated in its chromosome or with the aid of a replicableplasmid, a DNA sequence according to the invention. Preferably, thesequence comprises at least one of the genes of the sequence SEQ IDNO:1.

The invention also relates to any use of fragments of the sequence SEQID NO:1, or of fragments of the strand complementary to this sequence,of at least 15 base pairs, as primer for carrying out a PCR or as probefor detecting in vitro or inactivating in vivo genes of lactic bacteriainvolved in the biosynthesis of an EPS. This lower limit is setarbitrarily on account of the fact that small fragments which hybridizespecifically are generally 15-25 bp in length.

EXAMPLES

The present invention is described in greater detail below by means ofthe additional description which follows, which relates to examples ofobtaining DNA fragments, recombinant plasmids and transformed bacteriaaccording to the invention. These examples are preceded by a descriptionof the culture media. It is self-evident, however, that these examplesare given by way of illustration of the subject-matter of the invention,of which they in no way constitute a limitation. DNA manipulation andthe cloning and transformation of bacterial cells are, unless otherwisespecified, performed according to the protocols described in the work bySambrook et al. cited above. Percentages are given by weight exceptwhere otherwise stated.

Media: (add 1.5% of bacto-agar for a solid medium)

- M17 (Difco, U.S.A.): tryptone 0.5%, soytone 0.5%, hydrolysed meat0.5%, yeast extract 0.25%, ascorbic acid 0.05%, magnesium sulphate0.025%, disodium beta-glycerophosphate 1.9% and water.

- LM17: M17 medium comprising 1% of lactose.

- GM17: M17 medium comprising 1% of glucose.

- MSK: skimmed milk (10% reconstituted powder) comprising 0.1% of yeastextract.

- MAM: skimmed milk (10% reconstituted powder) comprising 10% of amixture of amino acids (495 mg/l Ala, 343 mg/l Arg, 682 mg/l Asp, 59mg/l Cys, 1229 mg/l Glu, 759 mg/l Gly, 153 mg/l His, 215 mg/l Iso, 470mg/l Leu, 565 mg/l Lys, 122 mg/l Met, 255 mg/l Phe, 436 mg/l Pro, 68mg/l Ser, 170 mg/l Thr, 61 mg/l Try, 304 mg/l Val adjusted to pH 5).

- HJL: tryptone 3%, beef extract 0.2%, yeast extract 1%, lactose 1% andKH₂ PO₄ pH 6.5 0.5%.

- Ruthenium red: 0.5% yeast extract, skimmed milk powder 10%, sucrose1%, agar 1.5% and 0.08 g/l of ruthenium red (see FR2,632,968).

Example I: cloning of a DNA fragment of S thermophilus strain Sfi6

I.1. Selection of an S. thermophilus strain producing EPS: the strainsof lactic bacteria from the Nestle collection are cultured in HJL liquidmedium, and dilutions thereof are plated out on ruthenium red solidmedium. Strains producing EPS remain white since the EPSs prevent thedye from staining their cell wall. In contrast, non-producing strainsstain red on account of the affinity of the dye for the peptidoglycan oftheir cell wall.

In this way, S. thermophilus strain Sfi6, which received the depositnumber CNCM I-1590 and which will be designated in the examples whichfollow by the expression "strain Sfi6", was selected from the lacticbacteria producing EPS.

I.2. Repeat structure of the EPS: the structure of the EPS produced bythe strain Sfi6 has been published by Doco et al. (Carbohyd. Res., 198,313-321, 1995). This EPS possesses the composition Glc:Gal:GalNac=1:2:1,and the tetrasaccharide repeat unit: ##STR4##

I.3. Mutagenesis with the transposon Tn916: the strain Sfi6 is renderedresistant to streptomycin by culturing it by repeated transfers in HJLmedium supplemented with contents increasing from 20 to 2000 μg/ml ofstreptomycin, and by then selecting the strains which become naturallyresistant.

The streptomycin-resistant strain Sfi6 and Enterococcus faecalis strainJH2-2, which possesses a plasmid pAM180 carrying the transposon Tn916(Tn916 is known to carry a tetracycline resistant gene; Gawron et al.,Nature, 300, 281-283, 1982) are conjugated. For this purpose, 1 ml of anovernight culture in M17 medium at 37° C. of E. faecalis strain JH2-2 ismixed with 10 ml of an overnight culture in HJL medium at 42° C. of thestrain Sfi6, the cells are centrifuged and resuspended in tubescomprising 100 μl of HJL medium, the suspension is applied to LM17 solidmedium which is incubated at 37° C. for 20 h, the cells are recovered byscraping and resuspended in tubes of 10 ml of HJL liquid medium, thetubes are incubated at 42° C. for 4 h, shaking them from time to time,and dilutions of the cultures are then plated out on solid LM17 mediumsupplemented with 2.5 μg/ml of tetracycline and 2000 μg/ml ofstreptomycin.

By carrying out 20 conjugations in parallel (independent mutations), itwas possible in this way to select 2×10⁴ tetracycline- andstreptomycin-resistant transconjugents.

I.4. Selection of mutants of the strain Sfi6 no longer producing EPSEPS(-)phenotype!: the resistant transconjugants are transferred ontoruthenium red solid medium supplemented with 2.5 μg/ml of tetracyclineand 2000 μg/ml of streptomycin. Approximately 10% of the transconjugentsform EPS(-) red colonies. Approximately 800 red colonies are thenselected and cultured overnight in microtitration plates comprising 200μl of HJL medium supplemented with 2.5 μg/ml of tetracycline. 100 μl ofthe HJL culture are then cultured in 1 ml of MSK milk. Approximately 25%of the red colonies tested display a stable EPS(-) phenotype in the milk(the milk is not thick and ropy, and analysis of the culture supernatantdoes not disclose any EPS). The other red colonies display an EPS(+)phenotype or recover the EPS(+) phenotype after several subcultures inthe milk.

In conclusion, the EPS(-) stable mutants have lost their capacity toproduce EPSs as a result of the integration of the transposon Tn916 intoa chromosomal gene involved in the biosynthesis of EPSs. In effect, theEPS(-) stable mutants can recover an EPS(+) phenotype when they arecultured in a growth medium lacking tetracycline (excision and loss ofthe transposon).

I.5 Characterization of EPS(-) stable mutants: approximately 100 stablemutants are analysed by the Southern blotting of a chromosomal DNApreparation from the mutants, digested with HindIII, and hybridizationof the Southern blot filter with the radioactive tetM gene (encodes atetracycline resistance) originating from the plasmid pIC182 (Hill etal., Applied and Env. Micro., 54, 1230-1236, 1988). Approximately 85% ofthe mutants analysed display an identical preponderant bandcorresponding to a locus called "locus A". For some of the othermutants, two further preponderant bands (locus B and C), correspondingto known loci involved in the biosynthesis of the cell wall (publicationin preparation), may be noted.

I.6 Characterization of the locus A: the chromosomal regions close tothe integrated transposon Tn916 may be isolated by reverse PCR. For thispurpose, 1 μg of a chromosomal DNA preparation from an arbitrarilychosen mutant (mutant No.1) is digested in a traditional manner withHindIII for 4h, the DNA is extracted with phenol/chloroform and dilutedin 720 μl of water, the diluted DNA is heated to 56° C. for 5 minutes,the DNA is cooled on ice, 80 μl of a 10-fold concentrated ligationbuffer and 5 units of a T4 ligase (Boehringer-Mannheim) are added to it,and it is incubated at 12° C. for 16 h, heated to 70° C. for 15 min toinactivate the ligase and then concentrated in a volume of 100 μl byseveral successive extractions in butanol. 10 μl of the ligationmixture, 100 pmol of primers, 15 mM dNTPs, 10 μl of buffer and 0.2 unitof Super-Taq polymerase (Stehlin GmbH) are then added into a PCR device.The nucleic acid primers are chosen on the basis of the known sequenceof the transposon Tn916.

Using the primers having the sequence SEQ ID NO:15 and SEQ ID NO:16, a1-kb fragment could be isolated by PCR. Furthermore, using the primersSEQ ID NO:17 and SEQ ID NO:18, a 4-kb fragment could be isolated (seethe sequence listing below).

A third, 0.8-kb fragment may also be isolated from the mutant No.1, bycarrying out a second reverse PCR from its chromosomal DNA digested withRsaI and using the primers having the sequences SEQ ID NO:18 and SEQ IDNO:19 (see the sequence listing below).

The 1-kb and 0.8-kb fragments were cloned into the linearized plasmidpGEMT (Promega, U.S.A.). Sequencing of these fragments by thedideoxynucleotide method (f-mol® DNA Sequencing System kit, Promega)shows two sequences which, on being matched up, cover three open readingframes (ORFs) corresponding to nucleotides 9933 to 11643 of the sequenceSEQ ID NO:1.

The 1-kb and 4-kb fragments were also used to screen a λ-ZAP Express(Stratagene, U.S.A.) library containing DNA fragments of the strainSfi6. For this purpose, according to the supplier's recommendations, aDNA preparation from the said mutant is partially digested with Sau3A,the fragments are separated by agarose gel electrophoresis, the bandscorresponding to 5- to 12-kb fragments are cut from the gel, and the DNAis eluted and then ligated to the λ-ZAP Express vector previouslydigested with BamHI. The ligation product is encapsidated in vitro usingthe GigagoldIII system (Stratagene), the phages are then mixed withEscherichia coli XL1Blue (Stratagene) according to the supplier'srecommendations and the mixture is then plated out on a Petri dish. Therecombinant plagues are then analysed by hybridization of their DNA,transferred onto a Hybond N membrane (Amersham Life Sciences, UK), withthe 1-kb and 4-kb fragments previously rendered radioactive (RandomPrimed DNA Labelling kit, Boehringer-Mannheim).

From 3000 recombinant plaques, approximately 20 positive plaques couldbe selected by hybridization, from which the λ-ZAP Express vectors werethen isolated, and the pCMV vectors containing a chromosomal insert werethereafter excised (see the recommendations of the supplier Stratagene).These recombinant vectors are called "pFS" in the examples which follow.

The chromosomal inserts of 11 pFS vectors were then sequenced (f-mol®DNA Sequencing System kit), these being the vectors pFS14, pFS15, pFS26,pFS30, pFS33, pFS49, pFS50, pFS65, pFS73, pFS80 and pFS86 (see FIG. 1.B)which comprise, respectively, fragments corresponding to the nucleotidesof the sequence SEQ ID NO:1, 9314-14602, 1-3159, 7988-11253, 1702-7991,1361-7229, 4400-8477, 648-7676, 5997-11253, 8474-13489, 3550-7229 and648-1702.

By matching up the nucleic acid sequences of the different chromosomalinserts, it was possible in this way to characterize a sequence of 15.2kb corresponding to the locus A of the strain Sfi6 (see FIG. 1.A).Nucleotides 648 to 15250 of this sequence of 15.2 kb are shown in thesequence SEQ ID NO:1.

I.7. Analysis of the sequence SEQ ID NO:1:

The sequence SEQ ID NO:1 comprises the whole of the eps operon of thestrain Sfi6. This sequence comprises 13 complete ORFs in the sameorientation, which are called eps A, B, C, D, E, F, G, H, I, J, K, L andM, (see FIG. 1.A). This sequence comprises, in addition, one completeORF at the 3' end of the sequence, which is encoded by the complementarystrand. This ORF, called orfZ, probably marks the end of the operon onaccount of its reverse orientation relative to the other ORFs.

Comparison of the amino acid sequences encoded by the first 13 ORFs withthose of the proteins present in the Swiss-Prot data bank, using thesoftwares FASTA, PEPPLOT and PILEUP from GCG-software, Wis., U.S.A.,enables the function of the 13 proteins encoded by the eps operon to bededuced. The results are presented below.

The epsA ORF (nucleotides 352-1803) codes for an EpsA protein (SEQ IDNO:2) having 26.4% identity with the LytR protein of Bacillus subtiliswhich is involved in the regulation of the autolysinN-acetylmuramoyl-L-alanine (Lazaveric et al., J. Gen. Microbiol., 138,1949-1961, 1992). Hence EpsA is probably a regulator protein for the epsoperon. Moreover, since a regulator ORF of an operon is generally foundupstream of the other ORFs, the epsA gene is probably the first gene ofthe eps operon. This is confirmed by the fact that a terminator is foundat nucleotides 230-252, a promoter at nucleotides 274-302 and a ribosomebinding site at nucleotides 340-345 of the sequence SEQ ID NO:1.

The epsB gene (nucleotides 1807-2535) codes for an EpsB protein (SEQ IDNO:3) having 67.5% identity with the CpsA protein of Streptococcusagalactiae and 30% identity with the CapC protein of Staphylococcusaureus (Rubens et al., Mol. Microbiol., 8, 843-885, 1993; Lin et al., J.Bacteriol., 176, 7005-7016, 1994). The precise function of these genesis still unknown, apart from the fact that they are essential for thesynthesis of the capsule which consists of polysaccharides coupled tothe phospholipids of the outer membrane of the bacteria.

The epsC gene (nucleotides 2547-3239) codes for an EpsC protein (SEQ IDNO:4) having 52% identity with the CpsB protein of Streptococcusagalactiae which is involved in the synthesis of the capsule (Rubens etal.). EpsC also has 23% identity, 49% similarity and a hydrophobicityprofile comparable to that of the CLD proteins of Salmonellatyphimurium, Salmonella enterica and Escherichia coli (Batchelor et al.,J. Bacteriol., 174, 5228-5236, 1992; Bastin et al., Mol. Microbiol., 7,725-734, 1993). It should be pointed out that the CLD proteins areinvolved in the control of the length of the polysaccharide chainsduring their biosynthesis.

The epsD gene (nucleotides 3249-3995) codes for an EpsD protein (SEQ IDNO:5) having 60.5% identity with the CpsC protein of Streptococcusagalactiae, having 34.5% identity with the CapA protein ofStaphylococcus aureus and having 33% identity with the ExoP protein ofRhizobium meliloti (Rubens et al., Lin et al.; Becker et al., Mol. Gen.Genet., 241, 367-379, 1993). The ExoP protein is a membrane proteinwhich is involved in the translocation of EPS and/or of EPS precursors.

The epsE gene (nucleotides 4051-4731) codes for an EpsE protein (SEDID-NO:6) displaying significant homologies with many proteins havinggalactosyltransferase activity (Rubens et al.). Hence this gene probablycodes for a galactosyltransferase.

It may be noted that the epsB, C, D and E genes of S. thermophilus Sfi6are similar to those of the operon of S. agalactiae comprising the cpsA,B, C and D genes (Rubens et al.,). Furthermore, they are organized inthe same way. Although the polysaccharides of the capsule and the EPS ofthe two strains are very different, this indicates that a chromosomalregion has probably been transferred between these two species.

The epsF gene (nucleotides 4898-5854) codes for an EpsF protein (SEQ IDNO:7) having, respectively, 24.5% and 23% identity with the CapH andCapM proteins of S. mutans which are probably involved asglycosyltransferases in the biosynthesis of the capsule (Lin et al.).

The epsG gene (nucleotides 6425-7540) codes for an EpsG protein (SEQ IDNO:8) having 20.5% identity and 50% similarity with theN-acetylglucosaminetransferase of Salmonella typhimurium LT2 which isinvolved in the biosynthesis of the LPS polysaccharide of the outermembrane (MacLachlan et al., J. Bacteriol., 173, 7151-7163, 1991). Sincean N-acetylglucosamine is not involved in the biosynthesis of the EPS ofthe strain Sfi6 (there is no acetylated glucose), the epsG gene probablycodes for a glucosyltransferase, an N-acetylgalactosyltransferase or anN-acetylglucosyltransferase having N-acetylglucosamine epimeraseactivity.

The epsH gene (nucleotides 7736-8212) codes for an EpsH protein (SEQ IDNO:9) having strong homologies with NodL-LacA-CysE acetyltransferases(Downie et al., Mol. Microbiol. 3, 1649-1651, 1989). Accordingly, theEpsH protein could be an acetyltransferase involved in the biosynthesisof the N-acetylgalactosamine of the EPS.

The epsI gene (nucleotides 8221-9192) codes for an EpsI protein (SEQ IDNO:10) having 24% identity with a protein, encoded by the RfbV ORF ofthe rfb cluster of Salmonella typhimurium, which is probably aglycosyltransferase (Jiang et al.; Liu et al., J. Bacteriol., 177,4084-4088, 1995).

The epsJ gene (nucleotides 9285-10364) codes for an EpsJ protein (SEQ IDNO:11) having 20% identity and a hydrophobicity profile comparable tothat of a protein of an ORF of the rfb cluster of Salmonella entericawhich is itself similar to a polymerase of the O antigen of group B andC2 salmonellae (Lee et al., J. Gen, Microbiol., 138, 1843-1855, 1992;Morona et al., J. Bacteriol. 176, 733-747, 1994). The epsJ gene couldhence encode an EPS polymerase which might polymerize thetetrasaccharide unit of the EPS.

The epsK gene (nucleotides 10392-11339) codes for an EpsK protein (SEQID NO: 12) having 18% identity and 42% similarity with the protein,encoded by the lipB gene of Neisseria meningitidis, which is involved inthe biosynthesis of the capsule by coupling polysaccharides to thephospholipids of the outer membrane (Frosch et al., Mol. Microbiol., 8,483-493, 1993). Given that the S. thermophilus bacteria do not have anouter membrane (Gram-positive), the epsK gene could hence encode anenzyme involved in the coupling of the EPSs to the phospholipids of thecell membrane which, in concert with an EPS transport molecule (probablyEpsC and EpsD) and an enzyme which detaches EPSs, might participate inthe transport of the EPS through the membrane (model in agreement withthe one put forward by Frosch et al.).

Moreover, it may be pointed out that the transposon Tn916 is integratedinto the epsK gene of the mutant No. 1 used to identify the eps operon(see point I.6 above), between nucleotides 10540-10541 of the sequenceSEQ ID NO:1.

The epsL gene (nucleotides 11302-12222) codes for an EpsL protein (SEQID NO:14) which does not display any homology with known proteins. Thefirst 38 nucleotides are covered by the 3' end of epsK, suggesting acoordinated expression of the two proteins and an activity of the EpsLprotein in the membrane transport of the EPS.

The epsM gene (nucleotides 12233-13651) codes for an EpsM protein (SEQID NO:13) which does not display any homology with known proteins of theSwiss-Prot data bank. This gene is definitely involved in thebiosynthesis of the EPS of the strain Sfi6, since there is not,upstream, a specific promoter for this gene.

The orfZ gene (13732-14305 on the complementary strand) is present inthe reverse orientation relative to the remainder of the ORFs of the epsoperon. Accordingly, it is probably not involved in the biosynthesis ofthe EPS of the strain Sfi6. Furthermore, it does not display anyhomology with known proteins of the Swiss-Prot data bank.

In conclusion, the chromosomal inserts isolated from the 11 pSF vectors(see point I.6 above) cover a chromosomal region of S. thermophilusstrain Sfi6 which is manifestly involved in the biosynthesis of the EPS.13 complete genes which comprise, upstream, a promoter delimiting thebeginning of the eps operon could thus be identified.

Example II: inactivation of the epsJ gene

The epsJ gene of the eps operon is inactivated by homologousrecombination in order to confirm its importance in the biosynthesis ofthe EPS.

For this purpose, a DraI-SalI fragment is isolated from plasmid pGEMTcontaining the 0.8-kb PCR fragment (see Example I.6 above) and ligatedinto the temperature-sensitive plasmid pSA3 (Dao et al., Appl. Environ.Microbiol., 49, 115-119, 1985) previously digested with EcoRV and SalI,the E. coli strain XL1-blue is transformed with the ligation product,transformants are selected, a recombinant plasmid is isolated, and S.thermophilus strain Sfi6 is transformed by electroporation with therecombinant plasmid by means of a method adapted from the one describedby Slos et al. (Appl. Environ. Microbiol., 57, 1333-1339, 1991). Thecells subjected to a discharge of 2.1 kV, 25 μF and 400Ω are resuspendedin 1 ml of HJL medium, which is incubated for 4 h at 37° C. (permissivetemperature), the cells are plated out on LM17 solid medium supplementedwith 2.5 μg/ml of erythromycin, which is incubated for 16 h at 37° C.,and the transformed colonies which survive are then selected. Theselected colonies are then incubated in 2 ml of HJL medium supplementedwith 2.5 μg/ml of erythromycin until the optical density at 600 nm(OD₆₀₀) of the culture reaches 0.2, the culture is subjected to 45° C.until the OD₆₀₀ reaches 1.0 (the plasmid no longer replicates), anddilutions of the culture are then plated out on solid LM17 mediumsupplemented with 2.5 μg/ml of erythromycin, which is incubated for 12 hat 45° C.

The colonies which survive have integrated the recombinant pSA3 plasmidinto the epsJ gene. This may be verified by Southern blotting of achromosomal DNA preparation of the surviving colonies, digested withEcoRI (cuts only once in pSA3), and hybridization of the Southern blotfilter with the abovementioned radioactive DraI-SalI fragment. Colonieswhich have integrated plasmid pSA3 display two bands on the Southernblot filter. Furthermore, colonies which have integrated the recombinantpSA3 plasmid into epsJ display an EPS(-) phenotype on ruthenium redsolid medium, and have lost their ropy character in MSK milk (seeExample I.4 above).

Example III: inactivation of the eps A, B, C, D, E, F, G, H, I, K, L andM genes

It was shown in Examples I and II that inactivation of the epsK and epsJgenes, by insertion of a transposon or of an integrative plasmid,interrupts EPS biosynthesis in the strain Sfi6.

Similarly, the other genes of the eps operon of the strain Sfi6 may beinactivated by homologous recombination, and an interruption of EPSbiosynthesis may thus be observed. For this purpose, a fragment of anORF originating from one of the 11 pFS vectors described in Example I.6above is amplified by PCR. It is cloned into plasmid pSA3, thentransformed and integrated into the strain Sfi6 under the sameconditions as those described in the previous-example.

Example IV: restoration of EPS production

pFS30 is cut with EcoRI, the fragments are separated, the 5.5-kbfragment is ligated to pFS14 previously digested with EcoRI, XL1-bluecells are transformed with the ligation product, transformed clonesdisplaying a correct orientation of the inserts are selected, a plasmidcalled pFS30-14 is isolated, a central EcoRI fragment of pFS65 isligated to pFS30-14 previously cut with EcoRI, XL1-blue cells aretransformed with the ligation product, and transformed clones displayinga correct orientation of the inserts are then selected. The resultantrecombinant plasmid, called pFS30-65-14, comprises nucleotides 1702 to14602 of the sequence SEQ ID NO: 1.

pFS30-65-14 is then cut with SalI and SmaI, the 12.9 kb fragment isseparated and ligated to pSA3 previously cut with EcoRV and SalI,XL1-blue cells are transformed with the ligation product, transformedclones are selected and recombinant pSA3 plasmids are isolated.

The S. thermophilus strain CNCM I-1292, deposited on 29th Mar. 1993, istransformed by electroporation with the recombinant pSA3 plasmids. ThisGram-positive strain displays under the microscope an appearance ofnon-flagellated cocci forming small chains, does not make spores, is afacultative anaerobe, does not produce any EPS and possesses in itsgenome 1000 bp corresponding to the 5' end of the eps operon. Therecombinant pSA3 plasmid can hence integrate into the genome of thestrain CNCM I-1292. Some of the transformed clones display an EPS(+)phenotype on ruthenium red solid medium, and a ropy character in MSKmilk.

Example V: restoration of EPS production

The chromosome of the strain Sfi6 is digested with enzymes which do notcut in the sequence SEQ ID NO:1 (BamHI, SalI, NruI, StuI), the digestionproduct is separated on agarose gel, the 15-25-kb bands are eluted andligated into pSA3 previously cut with a suitable restriction enzyme, theS. thermophilus strain CNCM I-1292 is transformed by electroporation,and transformants are then selected by transfer of the colonies onto afilter followed by hybridization of their DNA with the insert of pFS14previously made radioactive. Some of the transformed clones display anEPS(+) phenotype on ruthenium red solid medium, and a ropy character inMSK milk.

Example VI: modification of an EPS

The S. thermophilus strain CNCM I-1422, deposited on 18th May 1994, istransformed by electroporation with the recombinant pSA3 plasmid ofExample V. This Gram-positive strain displays under the microscope anappearance of non-flagellated cocci forming small chains, does not makespores, is a facultative anaerobe and produces an EPS having thecomposition Glc:Gal=2:2.

Example VII: modification of an EPS

The S. thermophilus strain CNCM I-1351, deposited on 5th Aug. 1993, istransformed by electroporation with the recombinant pSA3 plasmid ofExample V. This Gram-positive strain displays under the microscope anappearance of non-flagellated cocci forming small chains, does not makespores, is a facultative anaerobe and produces an EPS having thecomposition Glc:Gal:Rha=1:3:2.

Example VIII: modification of an EPS

The chromosomal DNA of the strain CNCM I-1590 is isolated by the methodof Slos et al. (Appl. Environ. Microbiol., 57, 1333-1339, 1991). The DNApreparation is digested with SacI and BamHI, the DNA fragments areseparated by electrophoresis on 0.7% agarose gel, the 12- to 16-kbfragments are eluted, and the DNA extracted is ligated to the vectorpJIM2279 (obtained from P. Renault, INRA, Jouy-en-Josas, Paris, France)previously digested with SacI and BamHI and then dephosphorylated.Lactococcus lactis strain MG1363 (J. Bacteriol., 154, 1-9, 1983),cultured on GM17 medium at 30° C., is transformed by the method of DeVos et al. (Gene, 85, 169-176, 1989). The transformed clones areselected by hybridization of the genomic DNA of the clones with one ofthe probes having the sequences SEQ ID NO: 15, 16, 17, 18 and 19. Among400 transformants, 6 positive clones are selected, one of whichcomprises a plasmid called pFS101 shown in FIG. 1.C.

To determine whether plasmid pFS101 is capable of inducing theproduction of recombinant EPS, L. lactis MG1363 is retransformed withpFS101 and plated out directly on ruthenium red solid medium. Forcomparison, L. lactis MG1363 is transformed with the plasmid pJIM2279and is then plated out directly on ruthenium red solid medium. Theresults show that all the colonies comprising pJIM2279 have a redphenotype (3000 EPS(-) colonies), while more than 99.5% of the coloniescomprising pFS101 have a white phenotype (800 EPS(+) colonies, apartfrom 2 colonies). Hence L. lactis strain MG1363 transformed with pFS101produces a recombinant EPS.

Production of the EPS of L. lactis strain MG1363 transformed with pFS101is brought about by culturing the organism in MAM medium, at a pH of5.5, at 30° C. with magnetic stirring at 60 rpm. The recombinant EPS isisolated by mixing the culture medium with 40% of trichloroacetic acid,centrifuging the mixture for 20 min at 8000 g, mixing the precipitatewith an equal volume of acetone, incubating the mixture at 4° C. for 12h, precipitating the mixture at 10,000 g for 1 h, suspending theprecipitate in water, adjusting the pH of the mixture to 7, dialysing itagainst water for 24 h, ultracentrifuging it at 100,000 g for 1 h,recovering the supernatant and then lyophilizing the supernatant. Forcomparison, L. lactis strain MG1363 transformed with pJIM2279 iscultured under the same conditions and the sugars are isolated in thesame manner.

The total amount of neutral sugars is determined by the method of Duboiset al. (Anal. Chem., 28, 350-356, 1956). The results show that thestrain transformed with pFS101 produces 10 mg/l of sugars, expressed asglucose equivalents, while the strain transformed with pJIM2279 producestraces of sugar (<1 mg/l).

The molecular weight of the recombinant EPS is estimated bychromatography on a Superose-6 (Pharmacia) gel filtration column whichis connected to the FPLC system (Pharmacia) previously calibrated withcommercial dextran (Sigma) of 2×10⁶ to 5×10³ daltons (Da). For thispurpose, 0.25 to 1 ml of a sample comprising 250 μg of neutral sugars isapplied to the column, which is eluted with a flow of 0.5 ml/min in 50mM phosphate buffer pH 7.2. For comparison, the sugars produced by thestrain transformed with pJIM2279 are separated in the same manner. Theresults presented in FIG. 2 show that the strain transformed withpJIM2279 produces a small amount of heterogeneous polysaccharides whoseorigin is definitely the cell wall (2-0.5×10⁶ Da; fractions 8-15 ) and alarge amount of low molecular weight oligosaccharides (mono-anddisaccharides; fractions 20-22). In contrast, the strain transformedwith pFS101 manifestly displays a recombinant EPS with a high molecularweight of approximately 2×10⁶ Da (fraction 9).

The sugar composition of the recombinant EPS is determined by gaschromatography by the method of Neeser et al. (Anal. Biochem., 142,58-67, 1984). The results show that the culture medium of the straintransformed with pFS101 comprises, in terms of molarity, a 1:3 ratio ofGlc:Gal. Traces of rhamnose originating from the cell wall may bedetected. In contrast, no GalNac is detected.

Hence the composition of the EPS produced by L. lactis strain MG1363transformed with pFS101 is different from that of the EPS produced bythe S. thermophilus strain CNCM I-1590. It may reasonably be estimatedthat the structure of the recombinant EPS is the same as that of the EPSof the strain CNCM I-1590, except for the fact that GalNac is replacedby a galactose.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14602 base pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 352..1803                                                       (D) OTHER INFORMATION: /product="epsA"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1807..2535                                                      (D) OTHER INFORMATION: /product="epsB"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 2547..3239                                                      (D) OTHER INFORMATION: /product="epsC"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3249..3995                                                      (D) OTHER INFORMATION: /product="epsD"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4051..4731                                                      (D) OTHER INFORMATION: /product="epsE"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 4898..5854                                                      (D) OTHER INFORMATION: /product="epsF"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 6425..7540                                                      (D) OTHER INFORMATION: /product="epsG"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 7736..8212                                                      (D) OTHER INFORMATION: /product="epsH"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 8221..9192                                                      (D) OTHER INFORMATION: /product="epsI"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 9285..10364                                                     (D) OTHER INFORMATION: /product="epsJ"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 10392..11339                                                    (D) OTHER INFORMATION: /product="epsK"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 11302..12222                                                    (D) OTHER INFORMATION: /product="CDS (eps L) covering CDS                     (epsk) on nucleotides 10392-11339"                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 12233..13651                                                    (D) OTHER INFORMATION: /product="epsM"                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 13732..14305                                                    (D) OTHER INFORMATION: /function="CDS on the                                  complementary strand"                                                         /product="orfz"                                                               (ix) FEATURE:                                                                 (A) NAME/KEY: terminator                                                      (B) LOCATION: 230..252                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: promoter                                                        (B) LOCATION: 274..302                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: RBS                                                             (B) LOCATION: 340..345                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TAGTTTGTAAAAGGACGCCATTTGGTCGTCCTTTTGTGTTGTAGCTAATATCTGTTCGAA60                GTGATAATAAGTTAAAATTTTTCAAACTACTAGAAAAAATAAAAATATTTGGAAGAAGAA120               GACTTATAATAAATAGGTAAATATCTGACAATTTAAAGTTTAACTACTAAAAATGTAAAA180               GATAGTTCACAATATAATGGAAAATGATATAAATTAAATGATTGATATCATAATGAAAAA240               CGTTTTCTTATTTTTTTGAAAAAAGAATGACAATTGAAATGAGGTTGTATTAATGTTATA300               ATAATAATAATAATGGGGAATACCTAATTTTAATTTTTAGGAGCAATTTATATGAGT357                  MetSer                                                                        TCGCGTACGAATCGTAAGCAAAAGCATACGAGTAATGGATCGTGGGGG405                           SerArgThrAsnArgLysGlnLysHisThrSerAsnGlySerTrpGly                              51015                                                                         ATGGTCAACGTTGGGTTGACCATCCTGTATGCTATTTTAGCATTGGTC453                           MetValAsnValGlyLeuThrIleLeuTyrAlaIleLeuAlaLeuVal                              202530                                                                        TTATTATTCACCATGTTCAATTATAATTTCCTATCCTTTAGGTTTTTG501                           LeuLeuPheThrMetPheAsnTyrAsnPheLeuSerPheArgPheLeu                              35404550                                                                      AACATCATTATCACCATTGGTTTGTTGGTAGTTCTTGCTATTAGCATC549                           AsnIleIleIleThrIleGlyLeuLeuValValLeuAlaIleSerIle                              556065                                                                        TTCCTTCAGAAGACTAAGAAATTACCACTAGTGACAACGGTTGTACTG597                           PheLeuGlnLysThrLysLysLeuProLeuValThrThrValValLeu                              707580                                                                        GTTATCTTCTCGCTAGTTTCTCTGGTTGGTATTTTTGGTTTTAAACAA645                           ValIlePheSerLeuValSerLeuValGlyIlePheGlyPheLysGln                              859095                                                                        ATGATTGACATCACTAACCGTATGAATCAGACAGCAGCATTTTCTGAA693                           MetIleAspIleThrAsnArgMetAsnGlnThrAlaAlaPheSerGlu                              100105110                                                                     GTAGAAATGAGCATCGTGGTTCCTAAGGAAAGTGACATCAAAGATGTG741                           ValGluMetSerIleValValProLysGluSerAspIleLysAspVal                              115120125130                                                                  AGCCAGCTTACTAGCGTACAGGCACCTACTAAGGTTGATAAGAACAAT789                           SerGlnLeuThrSerValGlnAlaProThrLysValAspLysAsnAsn                              135140145                                                                     ATCGAGATCTTGATGTCAGCTCTCAAAAAAGATAAAAAAGTTGATGTT837                           IleGluIleLeuMetSerAlaLeuLysLysAspLysLysValAspVal                              150155160                                                                     AAAGTTGATGATGTTGCCTCATATCAAGAAGCTTATGATAATCTCAAG885                           LysValAspAspValAlaSerTyrGlnGluAlaTyrAspAsnLeuLys                              165170175                                                                     TCTGGCAAATCTAAAGCTATGGTCTTGAGTGGCTCTTATGCTAGCCTA933                           SerGlyLysSerLysAlaMetValLeuSerGlySerTyrAlaSerLeu                              180185190                                                                     TTAGAGTCTGTCGATAGTAATTATGCTTCAAATCTAAAAACAATTTAT981                           LeuGluSerValAspSerAsnTyrAlaSerAsnLeuLysThrIleTyr                              195200205210                                                                  ACTTATAAAATTAAAAAGAAGAATAGCAACTCTGCAAACCAAGTAGAT1029                          ThrTyrLysIleLysLysLysAsnSerAsnSerAlaAsnGlnValAsp                              215220225                                                                     TCAAGAGTCTTCAATATTTATATTAGTGGTATTGATACCTACGGTCCG1077                          SerArgValPheAsnIleTyrIleSerGlyIleAspThrTyrGlyPro                              230235240                                                                     ATTTCAACAGTGTCACGTTCAGATGTCAATATCATTATGACAGTAAAC1125                          IleSerThrValSerArgSerAspValAsnIleIleMetThrValAsn                              245250255                                                                     ATGAATACACATAAGATTCTCTTGACGACTACTCCACGTGATGCATAC1173                          MetAsnThrHisLysIleLeuLeuThrThrThrProArgAspAlaTyr                              260265270                                                                     GTTAAGATTCCTGGTGGTGGGGCAGACCAGTATGATAAATTAACCCAC1221                          ValLysIleProGlyGlyGlyAlaAspGlnTyrAspLysLeuThrHis                              275280285290                                                                  GCAGGTATTTATGGCGTTGAAACATCTGAACAAACTCTAGAAGATCTT1269                          AlaGlyIleTyrGlyValGluThrSerGluGlnThrLeuGluAspLeu                              295300305                                                                     TATGGTATTAAGCTTGATTACTATGCACGAATTAACTTCACATCTTTC1317                          TyrGlyIleLysLeuAspTyrTyrAlaArgIleAsnPheThrSerPhe                              310315320                                                                     CTTAAGTTGATTGACCAACTTGGTGGTGTGACAGTCCATAATGATCAA1365                          LeuLysLeuIleAspGlnLeuGlyGlyValThrValHisAsnAspGln                              325330335                                                                     GCTTTCACACAAGAGAAGTTTGATTTCCCGGTTGGAGATATCCAAATG1413                          AlaPheThrGlnGluLysPheAspPheProValGlyAspIleGlnMet                              340345350                                                                     AATTCAGAGCAAGCACTTGGATTTGTTCGTGAACGCTATAATTTAGAT1461                          AsnSerGluGlnAlaLeuGlyPheValArgGluArgTyrAsnLeuAsp                              355360365370                                                                  GGCGGAGATAATGACCGTGGTAAAAACCAGGAGAAAGTTATTTCTGCG1509                          GlyGlyAspAsnAspArgGlyLysAsnGlnGluLysValIleSerAla                              375380385                                                                     ATTTTAAACAAGTTGGCTTCTCTAAAATCTGTATCAAACTTTACTTCA1557                          IleLeuAsnLysLeuAlaSerLeuLysSerValSerAsnPheThrSer                              390395400                                                                     ATCGTTAATAATCTCCAAGACTCTGTCCAAACGAATATGTCTTTGAAT1605                          IleValAsnAsnLeuGlnAspSerValGlnThrAsnMetSerLeuAsn                              405410415                                                                     ACCATTAACGCTTTGGCTAATACACAACTTGAATCAGGTTCTAAATTT1653                          ThrIleAsnAlaLeuAlaAsnThrGlnLeuGluSerGlySerLysPhe                              420425430                                                                     ACGGTGACTTCTCAAGCAGTAACAGGTACAGGTTCAACCGGACAATTG1701                          ThrValThrSerGlnAlaValThrGlyThrGlySerThrGlyGlnLeu                              435440445450                                                                  ATCTCTTATGCGATGCCAAATTCTAGTCTTTACATGATGAAACTAGAT1749                          IleSerTyrAlaMetProAsnSerSerLeuTyrMetMetLysLeuAsp                              455460465                                                                     AATTCGAGTGTGGAAAGTGCCTCTCAAGCTATCAAAAAATTGATGGAG1797                          AsnSerSerValGluSerAlaSerGlnAlaIleLysLysLeuMetGlu                              470475480                                                                     GAAAAATAAGTGATTGACGTTCACTCACATATTGTTTTTGATGTTGAT1845                          GluLysValIleAspValHisSerHisIleValPheAspValAsp                                 1510                                                                          GATGGTCCTGAAACTTTAGAAGAAAGTTTAGACCTCATTGGTGAAAGT1893                          AspGlyProGluThrLeuGluGluSerLeuAspLeuIleGlyGluSer                              152025                                                                        TACGCCCAGGGGGTACGTAAGATTGTTTCAACATCCCATCGTCGTAAG1941                          TyrAlaGlnGlyValArgLysIleValSerThrSerHisArgArgLys                              30354045                                                                      GGGATGTTTGAGACTCCAGAGGATAAAATTTTTGCCAACTTTAAAAAA1989                          GlyMetPheGluThrProGluAspLysIlePheAlaAsnPheLysLys                              505560                                                                        GTAAAAGCAGAAGCAGAAGCACTTTATCCAGACTTAACTATTTATTAT2037                          ValLysAlaGluAlaGluAlaLeuTyrProAspLeuThrIleTyrTyr                              657075                                                                        GGAGGTGAACTTTATTACACCTCAGACATTGTGGAGAAACTTGAAAAG2085                          GlyGlyGluLeuTyrTyrThrSerAspIleValGluLysLeuGluLys                              808590                                                                        AATCTCATTCCGCGCATGCACAACACTCAATTTGCTTTAATTGAGTTT2133                          AsnLeuIleProArgMetHisAsnThrGlnPheAlaLeuIleGluPhe                              95100105                                                                      AGTGCTCGCACATCTTGGAAAGAAATTCATAGTGGGCTTAGTAATGTT2181                          SerAlaArgThrSerTrpLysGluIleHisSerGlyLeuSerAsnVal                              110115120125                                                                  TTGAGAGCGGGGGTAACGCCTATTGTTGCTCATATTGAGCGCTATGAT2229                          LeuArgAlaGlyValThrProIleValAlaHisIleGluArgTyrAsp                              130135140                                                                     GCCCTCGAAGAAAATGCTGACCGTGTTCGAGAAATCATCAATATGGGC2277                          AlaLeuGluGluAsnAlaAspArgValArgGluIleIleAsnMetGly                              145150155                                                                     TGCTATACTCAAGTCAATAGCTCACATGTCCTCAAACCAAAGCTCTTT2325                          CysTyrThrGlnValAsnSerSerHisValLeuLysProLysLeuPhe                              160165170                                                                     GGAGATAAAGATAAAGTAAGAAAGAAACGTGTTCGCTTTTTCTTGGAG2373                          GlyAspLysAspLysValArgLysLysArgValArgPhePheLeuGlu                              175180185                                                                     AAAAATTTGGTTCATATGGTTGCTAGCGACATGCATAATCTTGGGCCG2421                          LysAsnLeuValHisMetValAlaSerAspMetHisAsnLeuGlyPro                              190195200205                                                                  AGACCACCATTTATGAAAGATGCTTATGAAATTGTTAAAAAGAACTAC2469                          ArgProProPheMetLysAspAlaTyrGluIleValLysLysAsnTyr                              210215220                                                                     GGCTCCAAACGTGCTAAGAATCTTTTTATTGAAAATCCCAAAACATTA2517                          GlySerLysArgAlaLysAsnLeuPheIleGluAsnProLysThrLeu                              225230235                                                                     CTAGAAAATCAATATTTATAGGAGATATTATGAATCAAGATAACACTAAA2567                        LeuGluAsnGlnTyrLeuMetAsnGlnAspAsnThrLys                                       24015                                                                         AGTGATGAAATCGACGTACTAGCATTGCTACATAAACTTTGGACGAAG2615                          SerAspGluIleAspValLeuAlaLeuLeuHisLysLeuTrpThrLys                              101520                                                                        AAGCTTTTGATTCTTTTCACAGCTTTTTATTTCGCTGTTTTCAGTTTC2663                          LysLeuLeuIleLeuPheThrAlaPheTyrPheAlaValPheSerPhe                              253035                                                                        TTAGGTACTTATTTCTTTATCCAACCAACATATACATCAACAACGCGT2711                          LeuGlyThrTyrPhePheIleGlnProThrTyrThrSerThrThrArg                              40455055                                                                      ATCTATGTTGTTAATCAGGCAACAGATAATAAGAATCTTTCTGCTCAA2759                          IleTyrValValAsnGlnAlaThrAspAsnLysAsnLeuSerAlaGln                              606570                                                                        GATTTGCAAGCTGGTACCTATTTGGCAAATGACTATAAAGAGATTATT2807                          AspLeuGlnAlaGlyThrTyrLeuAlaAsnAspTyrLysGluIleIle                              758085                                                                        GCATCAAATGATGTATTATCAGAAGTTATTAAAGATGAAAAATTGAAT2855                          AlaSerAsnAspValLeuSerGluValIleLysAspGluLysLeuAsn                              9095100                                                                       TTGAGTGAGGCAGAACTGTCTAAAATGGTTTCAGTTAATATTCCTACT2903                          LeuSerGluAlaGluLeuSerLysMetValSerValAsnIleProThr                              105110115                                                                     GATACTCGTCTTATTTCAATTTCTGTTAATGCTAAAACTGGTCAAGAT2951                          AspThrArgLeuIleSerIleSerValAsnAlaLysThrGlyGlnAsp                              120125130135                                                                  GCGCAAACACTTGCCAATAAGGTTCGTGAAGTTGCTTCAAAAAAAATC2999                          AlaGlnThrLeuAlaAsnLysValArgGluValAlaSerLysLysIle                              140145150                                                                     AAGAAGGTGACAAAAGTTGAAGATGTCACAACGCTCGAAGAAGCTAAA3047                          LysLysValThrLysValGluAspValThrThrLeuGluGluAlaLys                              155160165                                                                     TTGCCAGAGTCACCATCTTCACCAAATATCAAACTTAATGTGCTTCTT3095                          LeuProGluSerProSerSerProAsnIleLysLeuAsnValLeuLeu                              170175180                                                                     GGGGCAGTGCTTGGAGGATTCCTTGCAGTGGTTGGTGTATTGGTACGT3143                          GlyAlaValLeuGlyGlyPheLeuAlaValValGlyValLeuValArg                              185190195                                                                     GAAATCCTAGATGATCGTGTTCGCCGTCCAGAAGATGTGGAAGATGCC3191                          GluIleLeuAspAspArgValArgArgProGluAspValGluAspAla                              200205210215                                                                  CTTGGAATGACACTTCTTGGAATTGTCCCTGATACAGATAAAATTTAA3239                          LeuGlyMetThrLeuLeuGlyIleValProAspThrAspLysIle*                                220225230                                                                     GGAGAAGAAATGCCTTTATTAAAGTTAGTTAAATCAAAAGTAGACTTT3287                          MetProLeuLeuLysLeuValLysSerLysValAspPhe                                       1510                                                                          GCTAAAAAGACGGAAGAGTATTATAACGCTATTCGCACAAATATTCAA3335                          AlaLysLysThrGluGluTyrTyrAsnAlaIleArgThrAsnIleGln                              152025                                                                        TTTTCTGGTGCTCAGATGAAAGTGATTGCGATTAGCTCTGTTGAAGCT3383                          PheSerGlyAlaGlnMetLysValIleAlaIleSerSerValGluAla                              30354045                                                                      GGTGAAGGAAAATCAATGATATCTGTTAACTTGGCGATTTCATTTGCT3431                          GlyGluGlyLysSerMetIleSerValAsnLeuAlaIleSerPheAla                              505560                                                                        AGTGTTGGGCTCCGAACACTTCTGATTGATGCGGAAACGCGTAATTCT3479                          SerValGlyLeuArgThrLeuLeuIleAspAlaGluThrArgAsnSer                              657075                                                                        GTTTTGTCAGGTACATTTAAATCAAATGAGCCTTATAAAGGTCTTTCA3527                          ValLeuSerGlyThrPheLysSerAsnGluProTyrLysGlyLeuSer                              808590                                                                        AATTTCCTTTCAGGAAATGCCGATCTAAATGAAACGATTTGCCAAACT3575                          AsnPheLeuSerGlyAsnAlaAspLeuAsnGluThrIleCysGlnThr                              95100105                                                                      GATATTTCTGGTTTAGATGTTATTGCATCTGGTCCTGTTCCACCTAAT3623                          AspIleSerGlyLeuAspValIleAlaSerGlyProValProProAsn                              110115120125                                                                  CCAACAAGTCTTTTGCAAAATGATAATTTTAGACATTTGATGGAAGTT3671                          ProThrSerLeuLeuGlnAsnAspAsnPheArgHisLeuMetGluVal                              130135140                                                                     GCTCGTAGTTGTTATGATTATGTCATCATCGATACACCACCAGTTGGT3719                          AlaArgSerCysTyrAspTyrValIleIleAspThrProProValGly                              145150155                                                                     CTGGTTATTGATGCAGTTATTATTGCCCATCAGGCTGATGCCAGTCTT3767                          LeuValIleAspAlaValIleIleAlaHisGlnAlaAspAlaSerLeu                              160165170                                                                     TTGGTTACAGAAGCTGGGAAAATTAAACGTCGTTTCGTAACTAAGGCC3815                          LeuValThrGluAlaGlyLysIleLysArgArgPheValThrLysAla                              175180185                                                                     GTTGAACAATTGGTAGAAAGTGGTTCTCAGTTCTTAGGGGTCGTCCTT3863                          ValGluGlnLeuValGluSerGlySerGlnPheLeuGlyValValLeu                              190195200205                                                                  AATAAAGTTGACATGACAGTTGATAAATATGGATTTTATGGTTCTTAC3911                          AsnLysValAspMetThrValAspLysTyrGlyPheTyrGlySerTyr                              210215220                                                                     GGATCATATGGCGAGTATGGAAAAAAATCTGACCAAAAAGAAGGTCAT3959                          GlySerTyrGlyGluTyrGlyLysLysSerAspGlnLysGluGlyHis                              225230235                                                                     TCAAGAGCACATCGTCGTAGAAAAGTCGGTTGGAATTAACGCGTTA4005                            SerArgAlaHisArgArgArgLysValGlyTrpAsn                                          240245                                                                        GTGTGTTTTAAGATGTCGTTGGGAACGACAAGTGGAGGGAATGAGATGTCACAA4059                    MetSerGln                                                                     1                                                                             GCTAAAGAGGAAATTTCAGATGTTATGACTTATTCAGAGCTAACAAGT4107                          AlaLysGluGluIleSerAspValMetThrTyrSerGluLeuThrSer                              51015                                                                         CATAAGCCCAAAATTATTTATAGCTTGATTAAGCGGATTGGTGATATT4155                          HisLysProLysIleIleTyrSerLeuIleLysArgIleGlyAspIle                              20253035                                                                      TTGGTTAGTTCTATTGGTTTAATTATTTTGATACCGCTATTTTTGATA4203                          LeuValSerSerIleGlyLeuIleIleLeuIleProLeuPheLeuIle                              404550                                                                        GTTGCTTTGATCATGAAATGCTCTGAACCAACAGCACCTATATTTTTC4251                          ValAlaLeuIleMetLysCysSerGluProThrAlaProIlePhePhe                              556065                                                                        TCACATATTAGAAATGGTAAAAATGGCAAAAAGTTCAAAATGTATAAA4299                          SerHisIleArgAsnGlyLysAsnGlyLysLysPheLysMetTyrLys                              707580                                                                        TTTAGAACCATGTGTCAGGACGCAGAATCGATTTTGATGAAAGATACG4347                          PheArgThrMetCysGlnAspAlaGluSerIleLeuMetLysAspThr                              859095                                                                        GAACTTTTTGCAAAATTTAAGGCAAATGGTTATAAACTTGAAACGCAT4395                          GluLeuPheAlaLysPheLysAlaAsnGlyTyrLysLeuGluThrHis                              100105110115                                                                  GAAGATCCTAGAATTACAAAAATCGGTGGCATATTAAGGAAAACAAGT4443                          GluAspProArgIleThrLysIleGlyGlyIleLeuArgLysThrSer                              120125130                                                                     ATTGATGAATTGCCACAACTGATTAATGTTTTTTTAGGACAAATGTCA4491                          IleAspGluLeuProGlnLeuIleAsnValPheLeuGlyGlnMetSer                              135140145                                                                     TTAGTGGGTCCACGTCCACTACCAGATAGAGAAATCATTGAATACGGT4539                          LeuValGlyProArgProLeuProAspArgGluIleIleGluTyrGly                              150155160                                                                     GATAACCAAGAAAAATTTTTAAGCGTTAAACCAGGCATGACAGGATGG4587                          AspAsnGlnGluLysPheLeuSerValLysProGlyMetThrGlyTrp                              165170175                                                                     TGGCAAGTTTCAGGGAGAAGTACTATTGGGTATCCTGAGCGGTGTCAT4635                          TrpGlnValSerGlyArgSerThrIleGlyTyrProGluArgCysHis                              180185190195                                                                  CTTGAGCTTTATTATGTAGAAAAGTGTTGTTTTACTTTCGATGTTCTT4683                          LeuGluLeuTyrTyrValGluLysCysCysPheThrPheAspValLeu                              200205210                                                                     ATATTACTTAAGACAATTGGGATTGTTTTGAAGAGAGTTGGAGCGCGT4731                          IleLeuLeuLysThrIleGlyIleValLeuLysArgValGlyAlaArg                              215220225                                                                     TAGTACTGATGAAACAAAAATTATTATTGATAATAGAAGCGATGAGTGGTGGAGCCGGTC4791              GTCATGTACAAGACTTGATTAGTCATCTACCTCAAGAAAAATTTGATATTTATGTGATTT4851              ATTCAAATCATAGAACAAATCCTGTTTTTTGGAAAAAATAGTAACGATGAATGAG4906                   MetAsnGlu                                                                     1                                                                             CAAGTAACTTTTATTTTATGTGATTTTCTCGTAAGAGAAATTAAACCG4954                          GlnValThrPheIleLeuCysAspPheLeuValArgGluIleLysPro                              51015                                                                         AAATATGATTTGCTTGCTTATCAATTTATTTCTAAAAAGATTAAAGAA5002                          LysTyrAspLeuLeuAlaTyrGlnPheIleSerLysLysIleLysGlu                              20253035                                                                      ATCAAACCAGATATTGTACATTGTCACAGTTCAAAAGCTGGTGTTATT5050                          IleLysProAspIleValHisCysHisSerSerLysAlaGlyValIle                              404550                                                                        GGTCGTTTAGCTGCCAAAAGACGAGGTGTTAAAAAAATATTTTATACG5098                          GlyArgLeuAlaAlaLysArgArgGlyValLysLysIlePheTyrThr                              556065                                                                        CCACATGCTTATTCGTTTTTGGCACCTGAATTTAGTGGGAAGAAAAAG5146                          ProHisAlaTyrSerPheLeuAlaProGluPheSerGlyLysLysLys                              707580                                                                        TTTCTATTTGTTCAAATTGAAAAGTTTTTAAGCCGATTTGCGACAACT5194                          PheLeuPheValGlnIleGluLysPheLeuSerArgPheAlaThrThr                              859095                                                                        AAGATATTTTGTGTGTCAATAGCGGAAATGCAAGCTGCTCTTGAAGTA5242                          LysIlePheCysValSerIleAlaGluMetGlnAlaAlaLeuGluVal                              100105110115                                                                  AATCTAGATAAAACCGATAAGTTTCAGGTAATTTATAATGGTTTGCCA5290                          AsnLeuAspLysThrAspLysPheGlnValIleTyrAsnGlyLeuPro                              120125130                                                                     GAAATTGATTTACCAAGCAAAGAAACGATTCGGGCGCAATTAGGACTG5338                          GluIleAspLeuProSerLysGluThrIleArgAlaGlnLeuGlyLeu                              135140145                                                                     GAAAAGGCAGCAGTTGTTATAGGCAATAATGCAAAAATGTCGGAACAG5386                          GluLysAlaAlaValValIleGlyAsnAsnAlaLysMetSerGluGln                              150155160                                                                     AAAAATCCTATGTTTTTTATGGAAATTGCCCGAAAAATGATTAGACAA5434                          LysAsnProMetPhePheMetGluIleAlaArgLysMetIleArgGln                              165170175                                                                     AACGCAAATTGGCATTTTGTGTGGGTAGGTGATGGTCAGCTGATGCCA5482                          AsnAlaAsnTrpHisPheValTrpValGlyAspGlyGlnLeuMetPro                              180185190195                                                                  CTTTTTCAATCATTTATTAAGCAAAATGGACTAGAGGGAAATATCCAT5530                          LeuPheGlnSerPheIleLysGlnAsnGlyLeuGluGlyAsnIleHis                              200205210                                                                     TTGCTTGGCGAGCGTCCTGATAGTGAAATAGTTGTGACAGCCTATGAC5578                          LeuLeuGlyGluArgProAspSerGluIleValValThrAlaTyrAsp                              215220225                                                                     ATCTTCTTGACGACTTCCCAATATGAAGGTTTACCTTATGCACCAATT5626                          IlePheLeuThrThrSerGlnTyrGluGlyLeuProTyrAlaProIle                              230235240                                                                     GAAGCGATGCGAGCTGGTGTCCCGATTCTTGCGACAAAAGTTGTTGGC5674                          GluAlaMetArgAlaGlyValProIleLeuAlaThrLysValValGly                              245250255                                                                     AATAGTGAGCTTGTGATAGAGGGCAAAAATGGTTATTTGATTGACTTA5722                          AsnSerGluLeuValIleGluGlyLysAsnGlyTyrLeuIleAspLeu                              260265270275                                                                  GAGTGGTCAAAATCTGTCGAAGAAAAATTATATAAGGCAGCGAAAATA5770                          GluTrpSerLysSerValGluGluLysLeuTyrLysAlaAlaLysIle                              280285290                                                                     GATGCACAAATGATTAAAGCAGATTTTAGGCAAAGGTTTGCGATTGAT5818                          AspAlaGlnMetIleLysAlaAspPheArgGlnArgPheAlaIleAsp                              295300305                                                                     CAGATATTAAAGCAAATTGAAACAATTTATTTAGCTTGAATGAAGA5864                            GlnIleLeuLysGlnIleGluThrIleTyrLeuAla                                          310315                                                                        ATGAGGAGGCATAAATGCTGATTTTGAAATTAAAATTTCATCTTAATTGGTACACAAACG5924              AAAACCATTATTACACGTGAGTATTCGAAGACCTGGAAACGAGGCGATGAGCCGTATTAT5984              CCAGTGAACAATGATCGTAACAACAAACTCTATACTGCCTATAAGCGTCTTGCCGAGCAA6044              CAAGAGAATGTCATTTTCGGTGGACGTCTAGGTCACTACCGTTACTACGATATGCACCAG6104              GTAATTGGAGCTGCCTTGCAGTGTGTCAGAAATGAAGTGAAGTAAATCTTGATGAAGTTG6164              AATAACTTTAAGTAATTTTATACTTAATCCAATTGATGAAAATATTTTTGTATCGATTTA6224              TCTTCTGTAAGAAGAGTCCTAATCGTTTAAAAAATGTACAATTGAGTTTTTATATTTTTA6284              AATAAAGTTACTTTTAAGTCGTGTTATAGAATATACATGAATAGGTGTATTAGAAAATTT6344              ATTAATCTAATCCTCGAAAATAACTGACTGTAAGGAATCAAGTTGTGGAGTGTAAGTTGT6404              CAAATGGAGAGGAAAATAATATGAAAAAAATTTCAATTTTACACTTTTCC6454                        MetLysLysIleSerIleLeuHisPheSer                                                1510                                                                          CAAGTATCAGGCGGGGGAGTTGAAAAGTACATAAAATTATTTTTAAAG6502                          GlnValSerGlyGlyGlyValGluLysTyrIleLysLeuPheLeuLys                              152025                                                                        TATTCTGATGTGACAAAATTTAATAATTATTTAGTTGCACCTAATCTT6550                          TyrSerAspValThrLysPheAsnAsnTyrLeuValAlaProAsnLeu                              303540                                                                        GAAAATTATGACGAATTTAATGGATATTTAAAGATGTCTGTCAATTTT6598                          GluAsnTyrAspGluPheAsnGlyTyrLeuLysMetSerValAsnPhe                              455055                                                                        AATATGGAACAAACTTTTTCTCCGCTAAAAATATTCAAAAATGTCTTT6646                          AsnMetGluGlnThrPheSerProLeuLysIlePheLysAsnValPhe                              606570                                                                        TTTATTCGTAGTGTACTCAAAAAAATAAACCCAGATATAGTATACCTA6694                          PheIleArgSerValLeuLysLysIleAsnProAspIleValTyrLeu                              75808590                                                                      CATAGTACATTTGCAGGTGTCGTAGGTCGTATTGCTTCAATAGGTTTG6742                          HisSerThrPheAlaGlyValValGlyArgIleAlaSerIleGlyLeu                              95100105                                                                      CCAACAAAAGTAGTATACAATCCTCACGGATGGTCCTTCAAAATGGAC6790                          ProThrLysValValTyrAsnProHisGlyTrpSerPheLysMetAsp                              110115120                                                                     AACAGCTATTTGAAAAAGCTTATTTTTAAATTAATCGAATTTTCTTTA6838                          AsnSerTyrLeuLysLysLeuIlePheLysLeuIleGluPheSerLeu                              125130135                                                                     TCTTTTTTAACTGATAAGTTTATTTTAATTTCGGAATCTGAGTATATT6886                          SerPheLeuThrAspLysPheIleLeuIleSerGluSerGluTyrIle                              140145150                                                                     TTGGCTAACCATATTTCATTTAATAAAAGCAAGTTTTCACTAATTAAT6934                          LeuAlaAsnHisIleSerPheAsnLysSerLysPheSerLeuIleAsn                              155160165170                                                                  AATGGTGTTGAAGTGATTACAGGGGATTCAAGAAATGAGATAGAAGAG6982                          AsnGlyValGluValIleThrGlyAspSerArgAsnGluIleGluGlu                              175180185                                                                     ATATTTCCAAATGAGGATTTTATAATTGGCATGGTTGGCAGACTAAGC7030                          IlePheProAsnGluAspPheIleIleGlyMetValGlyArgLeuSer                              190195200                                                                     CCACCCAAAGAGTTTTTCTTTTTTATTGATTTTGCAAAAAAAATATTA7078                          ProProLysGluPhePhePhePheIleAspPheAlaLysLysIleLeu                              205210215                                                                     CAAATTCGAAACGATACCAATTTTATTATCGTGGGTGATGGAGAGTTA7126                          GlnIleArgAsnAspThrAsnPheIleIleValGlyAspGlyGluLeu                              220225230                                                                     CGAAGTGAAATAGAAAGAATGATACTAGATAATGGGTTAGGAGATAAA7174                          ArgSerGluIleGluArgMetIleLeuAspAsnGlyLeuGlyAspLys                              235240245250                                                                  ATCTATATTACTGGGTGGGTTGATAATCCGAGAAACTATATAGAGAAG7222                          IleTyrIleThrGlyTrpValAspAsnProArgAsnTyrIleGluLys                              255260265                                                                     TTTGATCAAGCTATTCTGTTTTCTAGATGGGAGGGTCTTAGCCTAACG7270                          PheAspGlnAlaIleLeuPheSerArgTrpGluGlyLeuSerLeuThr                              270275280                                                                     ATTGCGGAATATATGTCTCAGAAGAAAACAATTTTAGCAACAAATATT7318                          IleAlaGluTyrMetSerGlnLysLysThrIleLeuAlaThrAsnIle                              285290295                                                                     GGTGGCATTAATGATTTAATCACTGATGGTGAAACAGGAATGCTGATT7366                          GlyGlyIleAsnAspLeuIleThrAspGlyGluThrGlyMetLeuIle                              300305310                                                                     GAAGTTGGAGACTTGAATTCAGCAGTATCTAAATCTTTCGAGCTAAGA7414                          GluValGlyAspLeuAsnSerAlaValSerLysSerPheGluLeuArg                              315320325330                                                                  AATAATAAAGAGGTTTCGAATCAATTAGCGAATAACGCTTATAATAAA7462                          AsnAsnLysGluValSerAsnGlnLeuAlaAsnAsnAlaTyrAsnLys                              335340345                                                                     GTTGTTGAACAGTTTTCGATTGAAAAACAGATGGCTGAGATAGAAAGT7510                          ValValGluGlnPheSerIleGluLysGlnMetAlaGluIleGluSer                              350355360                                                                     TTATTTATAGAGATGTGTAACAATGAGAAATAGAGACTTAAAGAAAATAC7560                        LeuPheIleGluMetCysAsnAsnGluLys                                                365370                                                                        AGGTTATTTGATTGACTTAGAGTGGTCAAAATCTGTCGAAGAAAAATTATATAAGGCAGC7620              GAAAATGGATGCACAAATGATTAAAGCAGATTTTAGGCAAAGGTTTGCGATTGATCAGAT7680              GTTAAAGCAAATTAAAACAATTTATTTAGCTTGAATGAAGAAAGAGGAGGCATAAATG7738                Met                                                                           1                                                                             CTGATTTTGAAATTAAAATTTCATCTTAAATCGTTATTCCTTAAATGG7786                          LeuIleLeuLysLeuLysPheHisLeuLysSerLeuPheLeuLysTrp                              51015                                                                         ATTTATCGATTACTTTATCTAAAAAAGTTTCAGTTTGGTGCACGCTTG7834                          IleTyrArgLeuLeuTyrLeuLysLysPheGlnPheGlyAlaArgLeu                              202530                                                                        ACGTTTCGAGATGGGTTTCATTTGTTAATTGAAAAATCTGGGAAAGTT7882                          ThrPheArgAspGlyPheHisLeuLeuIleGluLysSerGlyLysVal                              354045                                                                        ATCATCGGGAATCATGTTTTTTTTAATAACTTTTGTTCAATTAATGCC7930                          IleIleGlyAsnHisValPhePheAsnAsnPheCysSerIleAsnAla                              50556065                                                                      ATGTTATCAGTAACGATTGGTGATGACTGTATTTTTGGTGAAAACGTT7978                          MetLeuSerValThrIleGlyAspAspCysIlePheGlyGluAsnVal                              707580                                                                        AAAATTTATGATCACAATCATTGTTATCAAAATAAAAGTCAACCTATT8026                          LysIleTyrAspHisAsnHisCysTyrGlnAsnLysSerGlnProIle                              859095                                                                        TCAAAACAAGGTTTTTCAACTGCTGCTATCCAGATTGGTCGTAACTGT8074                          SerLysGlnGlyPheSerThrAlaAlaIleGlnIleGlyArgAsnCys                              100105110                                                                     TGGATAGGTAGTCAAGTGACGATTTTAAAAGGTGTAACCATAGGTGAT8122                          TrpIleGlySerGlnValThrIleLeuLysGlyValThrIleGlyAsp                              115120125                                                                     AATAGTATCATTGGTGCTGGTGTGGTAGTTTATCAAGATGTGCCAGAA8170                          AsnSerIleIleGlyAlaGlyValValValTyrGlnAspValProGlu                              130135140145                                                                  AATTCGATTGTTTTATCTAATGGAGAAATTAGAAAGCGTGGC8212                                AsnSerIleValLeuSerAsnGlyGluIleArgLysArgGly                                    150155                                                                        TAATTAAAATGTATCTTAAAAGTCTAATCTCTATTGTTATTCCAGTATAT8262                        MetTyrLeuLysSerLeuIleSerIleValIleProValTyr                                    1510                                                                          AATGTAGAGAAATATTTAGAAAAATGTTTGCAATCTGTTCAAAATCAG8310                          AsnValGluLysTyrLeuGluLysCysLeuGlnSerValGlnAsnGln                              15202530                                                                      ACTTACAATAATTTTGAAGTGATTTTAGTGAATGATGGCTCAACCGAT8358                          ThrTyrAsnAsnPheGluValIleLeuValAsnAspGlySerThrAsp                              354045                                                                        TCATCACTTTCAATATGCGAAAAATTTGTTAATCAGGATAAAAGATTT8406                          SerSerLeuSerIleCysGluLysPheValAsnGlnAspLysArgPhe                              505560                                                                        TCTGTTTTTTCTAAAGAAAATGGTGGTATGTCATCTGCACGAAATTTT8454                          SerValPheSerLysGluAsnGlyGlyMetSerSerAlaArgAsnPhe                              657075                                                                        GGAATTAAAAAGGCTAAAGGATCGTTTATCACATTTGTAGATAGTGAT8502                          GlyIleLysLysAlaLysGlySerPheIleThrPheValAspSerAsp                              808590                                                                        GACTACATAGTAAAAGATTATCTTTCTCATTTGGTAGCTGGGATAAAA8550                          AspTyrIleValLysAspTyrLeuSerHisLeuValAlaGlyIleLys                              95100105110                                                                   AGTGAGACCTCTATAGTTTGTTCAAAGTTTTTTCTTGTAGATGAAAAA8598                          SerGluThrSerIleValCysSerLysPhePheLeuValAspGluLys                              115120125                                                                     GGAAGTTTATTGACTAAAAAAGAGGCACCTAAAAAGAAATCAGAAGTC8646                          GlySerLeuLeuThrLysLysGluAlaProLysLysLysSerGluVal                              130135140                                                                     GTTTCAATTGAGGAAAGTATTAAAATTCTTCTGTTGCAACAAAATGGC8694                          ValSerIleGluGluSerIleLysIleLeuLeuLeuGlnGlnAsnGly                              145150155                                                                     TATGATCTCGCTGTCTGGGGAAAATTATACCCCGTTTCTTTCTTTGAA8742                          TyrAspLeuAlaValTrpGlyLysLeuTyrProValSerPhePheGlu                              160165170                                                                     ACAATTTCTTTCCCAGAAGGAAAACTTTACGAAGATATGGGAACAACT8790                          ThrIleSerPheProGluGlyLysLeuTyrGluAspMetGlyThrThr                              175180185190                                                                  TACAAATTACTAAAATTGGCAAGTGAAGTGGTCTTCTTGGATGCGTAT8838                          TyrLysLeuLeuLysLeuAlaSerGluValValPheLeuAspAlaTyr                              195200205                                                                     GATTATGCCTACGTACAGCGACCTAATAGTATCATGAATAGTTCTTTT8886                          AspTyrAlaTyrValGlnArgProAsnSerIleMetAsnSerSerPhe                              210215220                                                                     AATTTGAAAAAGTTGGATATAATAGAAATGGTTCATGAAATGGAAAAC8934                          AsnLeuLysLysLeuAspIleIleGluMetValHisGluMetGluAsn                              225230235                                                                     GATATATTAGCACAGTTTCCAAATTTAGCATTATATGTTAAGAATCGA8982                          AspIleLeuAlaGlnPheProAsnLeuAlaLeuTyrValLysAsnArg                              240245250                                                                     GCATTTGCCGCGGAAGTGAAAATCTTTTTAGAGATTCCAAAAGAAAAA9030                          AlaPheAlaAlaGluValLysIlePheLeuGluIleProLysGluLys                              255260265270                                                                  GAATTTGAGCAAGCGCAAAAGCAACTTTGGCATGATATCAAAAAGAAT9078                          GluPheGluGlnAlaGlnLysGlnLeuTrpHisAspIleLysLysAsn                              275280285                                                                     AGAAAAGCACCATTTATGACAAAAGGTGCTAGATTGAAGAATAGGCTC9126                          ArgLysAlaProPheMetThrLysGlyAlaArgLeuLysAsnArgLeu                              290295300                                                                     GGAGCTAGTCTGTCGTTTTTAGGTAAATCTTTATTTTTGACTATTGGG9174                          GlyAlaSerLeuSerPheLeuGlyLysSerLeuPheLeuThrIleGly                              305310315                                                                     AAGCAGTTAGTAGATAGATAATGATATTGAAAGCGATACGATACAATC9222                          LysGlnLeuValAspArg                                                            320                                                                           GTAAACTTCTTTTGGTGTTGACTAGGAGTTAGCTTGAAATTTGAATATAAAGGAAGCAAC9282              ACATGGTAATTTATTTTTTACTTTTCCCGATGATCGCAATGATTTAT9329                           MetValIleTyrPheLeuLeuPheProMetIleAlaMetIleTyr                                 151015                                                                        TTAATGACATTGCTCTTACGGCAAAAAGCACAAATCCAAAAAACGATT9377                          LeuMetThrLeuLeuLeuArgGlnLysAlaGlnIleGlnLysThrIle                              202530                                                                        TTTTGTGTTCTTACGTTTGGTACACTAGGCTTTATTTCAGCAAGTCGT9425                          PheCysValLeuThrPheGlyThrLeuGlyPheIleSerAlaSerArg                              354045                                                                        GCATCAAGTGTTGGGACGGACGTTACTTTATACGAAAATATTTTTAAA9473                          AlaSerSerValGlyThrAspValThrLeuTyrGluAsnIlePheLys                              505560                                                                        TCTATAAATTACGGGATAAGTGCTGAGAATAATTGGGGATACGTCATC9521                          SerIleAsnTyrGlyIleSerAlaGluAsnAsnTrpGlyTyrValIle                              657075                                                                        TATAACAAGCTGATTGGTAGTGTATTTGGCTATACAGGACATGAAATC9569                          TyrAsnLysLeuIleGlySerValPheGlyTyrThrGlyHisGluIle                              80859095                                                                      ACGGCCGCTAATTCAGTTTTGATTACAATACTTATTGGTATTTTTATT9617                          ThrAlaAlaAsnSerValLeuIleThrIleLeuIleGlyIlePheIle                              100105110                                                                     TGGAAAGTAGCGGAACATTATTTTGTTGCGACGTTTTTATACATTAGC9665                          TrpLysValAlaGluHisTyrPheValAlaThrPheLeuTyrIleSer                              115120125                                                                     TTGTTTTATTATGCTACAAGTTTTAATATTTCAAGACAATTTATTGCC9713                          LeuPheTyrTyrAlaThrSerPheAsnIleSerArgGlnPheIleAla                              130135140                                                                     ATGGGGCTTGTATTGGTAGCAATTTCTTTTGCTTTAGATAAAAAGGTT9761                          MetGlyLeuValLeuValAlaIleSerPheAlaLeuAspLysLysVal                              145150155                                                                     ATGCCTTGGTTTATCTTGACAGTTTTGGCTACCTTATTTCATGCGACA9809                          MetProTrpPheIleLeuThrValLeuAlaThrLeuPheHisAlaThr                              160165170175                                                                  GCAATCGTTGCTTTTCCTGTCTATTGGCTTACAAAAGTACATTGGGAT9857                          AlaIleValAlaPheProValTyrTrpLeuThrLysValHisTrpAsp                              180185190                                                                     GTGAAAAAGACATTAAGTATTTTTCCAATCACGATTTTTGCAAGTTTT9905                          ValLysLysThrLeuSerIlePheProIleThrIlePheAlaSerPhe                              195200205                                                                     ATTTTTGATGCTATTTTAAACATTTTTGTACGTTTTTTCCCACATTAT9953                          IlePheAspAlaIleLeuAsnIlePheValArgPhePheProHisTyr                              210215220                                                                     GAGATGTATATCACTGGAACACAATTTAATATTTCAGATCAGGGGCAG10001                         GluMetTyrIleThrGlyThrGlnPheAsnIleSerAspGlnGlyGln                              225230235                                                                     GGACGTGTGGTTTTGGTCAAAATATTTATCTTGCTCATTTTGTTTACT10049                         GlyArgValValLeuValLysIlePheIleLeuLeuIleLeuPheThr                              240245250255                                                                  TTATTCTTGTTTTATAAAAAAAGCTATGCTTTGATTTCTGAATGTCAT10097                         LeuPheLeuPheTyrLysLysSerTyrAlaLeuIleSerGluCysHis                              260265270                                                                     CAAAGTTTGATAGCTTTGACAACCGTTGGATTAAGTATCGGTATTGTA10145                         GlnSerLeuIleAlaLeuThrThrValGlyLeuSerIleGlyIleVal                              275280285                                                                     TTTTATAATAATATTTTACTCAATAGAATAGAAATGTTTTATTCAATT10193                         PheTyrAsnAsnIleLeuLeuAsnArgIleGluMetPheTyrSerIle                              290295300                                                                     TTAAGCATCGTATTTATTCCAATTGCTATAGATTACATTAGTTTGAAA10241                         LeuSerIleValPheIleProIleAlaIleAspTyrIleSerLeuLys                              305310315                                                                     TTTAAACAAAAAGATGCTGTGCGACTAATGCTGACGATAGGTATTTTG10289                         PheLysGlnLysAspAlaValArgLeuMetLeuThrIleGlyIleLeu                              320325330335                                                                  TTAATTACACTTGTGCCTTACTATATACAGGTTAGCGGTAATTATTCA10337                         LeuIleThrLeuValProTyrTyrIleGlnValSerGlyAsnTyrSer                              340345350                                                                     GGAATATTGCCTTATGTTATTCAACAATAAAAAATAAAGTTTAGAGA10384                          GlyIleLeuProTyrValIleGlnGln                                                   355360                                                                        GGAAATAATGGAGGATAGAAAGAAACAAGTAATTTTGATACTATCCCAC10433                        MetGluAspArgLysLysGlnValIleLeuIleLeuSerHis                                    1510                                                                          AGAAATACTCTCGCTCTAAAATCAACAATAGAGCTTTTGGATTCTCAA10481                         ArgAsnThrLeuAlaLeuLysSerThrIleGluLeuLeuAspSerGln                              15202530                                                                      TACTTTGATTTCTTTCTTCATATAGATAAAAAAAGTAGAATTCAAGAT10529                         TyrPheAspPhePheLeuHisIleAspLysLysSerArgIleGlnAsp                              354045                                                                        TTTTTTTATTTAAAAAAAATTACAAAATTCTCCACTATTCATTTTTCA10577                         PhePheTyrLeuLysLysIleThrLysPheSerThrIleHisPheSer                              505560                                                                        GAAAGAAAAAATGTACATTGGGGAGGTTTTTCTATGGTAGAAGCAATG10625                         GluArgLysAsnValHisTrpGlyGlyPheSerMetValGluAlaMet                              657075                                                                        TTTGCGCTATTAGAATGTGCACGTGATACAGGAGAATATTCTTATTTT10673                         PheAlaLeuLeuGluCysAlaArgAspThrGlyGluTyrSerTyrPhe                              808590                                                                        CATTTTTTATCTGGAGATGATATGCCAATCAAAGATAATGAAATAGTA10721                         HisPheLeuSerGlyAspAspMetProIleLysAspAsnGluIleVal                              95100105110                                                                   TTTAATTTTTTTGAAAATAGTTATCCTAAAAATTTTATTGATATTCTA10769                         PheAsnPhePheGluAsnSerTyrProLysAsnPheIleAspIleLeu                              115120125                                                                     GATTTTGAAAATGTCAATAAAAATTCATATTTCTACGAACCCCCTGAG10817                         AspPheGluAsnValAsnLysAsnSerTyrPheTyrGluProProGlu                              130135140                                                                     ATGATAGAGGAGAGAGTGAAGTACTACTATCCTCATATGGATATTCTA10865                         MetIleGluGluArgValLysTyrTyrTyrProHisMetAspIleLeu                              145150155                                                                     AACAGAAAAGGAACAAATTTCATAGGGAAAAAACTAATTTATCTACAA10913                         AsnArgLysGlyThrAsnPheIleGlyLysLysLeuIleTyrLeuGln                              160165170                                                                     AAATTGTTGAAAGTTAATCGCTTGAAAAATAGAGAGATAGAAATTTTC10961                         LysLeuLeuLysValAsnArgLeuLysAsnArgGluIleGluIlePhe                              175180185190                                                                  AAGGGTCATCAATGGTGTAGTTTGACAAATCAATTTGTAGATATTTTA11009                         LysGlyHisGlnTrpCysSerLeuThrAsnGlnPheValAspIleLeu                              195200205                                                                     TTGGATAAAGAGGAAAGAAGAGTAGGTAAGTCTTATTTTTCATCTAGT11057                         LeuAspLysGluGluArgArgValGlyLysSerTyrPheSerSerSer                              210215220                                                                     TTAATACCAGATGAATGTTATTTTCAAACGTTTGCTATGATAAAAAAA11105                         LeuIleProAspGluCysTyrPheGlnThrPheAlaMetIleLysLys                              225230235                                                                     GTTGAAATTTATCAACAGAAAAATATGTCAGCACGCTTAATTGATTGG11153                         ValGluIleTyrGlnGlnLysAsnMetSerAlaArgLeuIleAspTrp                              240245250                                                                     ACAAGAGGGAAACCATATATTTGGCGACAGGATGATTTTTTTGAAATT11201                         ThrArgGlyLysProTyrIleTrpArgGlnAspAspPhePheGluIle                              255260265270                                                                  ATGAATGATAAAGATTCAATGTTTTCTAGGAAGTTTGATGAAAATGTA11249                         MetAsnAspLysAspSerMetPheSerArgLysPheAspGluAsnVal                              275280285                                                                     GATCGTAAAATAATTGAAGAAATTTATATAAAAATAAGAGGAAGAAGT11297                         AspArgLysIleIleGluGluIleTyrIleLysIleArgGlyArgSer                              290295300                                                                     ACTGATGAAGCAAATAAAATCAAAGATAAGAGATTTACAAAA11339                               ThrAspGluAlaAsnLysIleLysAspLysArgPheThrLys                                    305310315                                                                     TAATTTTACCTATGTTTTTGGAAAGAAAACTTTTCTTGGAAGGGGAGAAGCGATTATCAT11399             AGATGAACCTGAGCATGGAAATTTGGGAGATCAAGCAATTGCTTTTGCAGAAAATCAATT11459             TTTAGTAAATCATGTATCAGTACGAGATGTAGAACATCTTATAGAAAGCAAAACTATTTC11519             AGAAATAAAATCTATAAAAAAAAATATTGGAAAAAAAGAATTAGTTTTTTTTCATGGGGG11579             AGGAAATTTCGGGACACTTTATCTAAAGTATGAGCGCATTAGAAGATTGGCAGTATCAAA11639             GCTTCCCTTTAATAAAATGATTCTATTTCCTCAGTCAATTTCATTTGAAGATAGTAGGTT11699             TGGTCAGAAGCAGCTGAATAAAAGTAAAAAAATATACAGTCAAAATACAAATTTTATTTT11759             GACTGCAAGAGAACCAAAATCTTATGGTTTAATGAAGAAATGTTTTCCATATAACAAAGT11819             AATCTTGACACCGGATATCGTGCTCTCATTTAAATTTGAAGTCACCATTTCTGATACGCA11879             TATTGGGAAAGAAAAGGATAGTGTTATAACTTATGAAAATCGTCAACACTATCTTGAGAT11939             AAAGTGGGATGAAATTGCGCAGCATGAGGTCGCCTTAACTGATAGATTACATGGTATGAT11999             TTTTTCATATATCACAGGCACACCATGTGTTGTTTTGGCTAATAATAATCATAAAATTGA12059             AGGAACATACAAACATTGGTTGAATGAAGTCAACTATATTCGTTTTATTGAAAATCCGAC12119             TGTTGAAAATATTTTAGATGCAATCAATGACTTAAAGCAAATCGAACCTCACTATATTGA12179             TTTATCTGATAAATTTCAACCACTAATTGATGCGATAAAAGGGTAAAGGTTTAATG12235                 Met                                                                           1                                                                             AATAAATATAAAAAACTACTATCCAACTCTCTTGTTTTCACGATAGGA12283                         AsnLysTyrLysLysLeuLeuSerAsnSerLeuValPheThrIleGly                              51015                                                                         AACTTAGGCAGCAAACTGTTAGTCTTTTTACTCGTACCGCTCTACACC12331                         AsnLeuGlySerLysLeuLeuValPheLeuLeuValProLeuTyrThr                              202530                                                                        TATGCGATGACACCGCAAGAGTATGGTATGGCAGACTTATATCAAACA12379                         TyrAlaMetThrProGlnGluTyrGlyMetAlaAspLeuTyrGlnThr                              354045                                                                        ACAGCAAATCTACTTTTGCCATTAATTACAATGAATGTATTTGATGCA12427                         ThrAlaAsnLeuLeuLeuProLeuIleThrMetAsnValPheAspAla                              50556065                                                                      ACTTTACGTTTTGCTATGGAAAAGTCAATGACAAAAGAGAGTGTGTTA12475                         ThrLeuArgPheAlaMetGluLysSerMetThrLysGluSerValLeu                              707580                                                                        ACAAATTCTCTTGTGGTTTGGTGTTTTAGCGCGGTGTTCACTTGTTTG12523                         ThrAsnSerLeuValValTrpCysPheSerAlaValPheThrCysLeu                              859095                                                                        GGCGCTTGTATTATCTATGCGTTGAACTTGAGTAATAAATGGTATTTA12571                         GlyAlaCysIleIleTyrAlaLeuAsnLeuSerAsnLysTrpTyrLeu                              100105110                                                                     GCTTTACTTTTAACCTTCAACTTATTTCAAGGTGGACAAAGTATATTA12619                         AlaLeuLeuLeuThrPheAsnLeuPheGlnGlyGlyGlnSerIleLeu                              115120125                                                                     AGCCAGTATGCTAGAGGTATAGGAAAGTCGAAAATATTTGCAGCTGGC12667                         SerGlnTyrAlaArgGlyIleGlyLysSerLysIlePheAlaAlaGly                              130135140145                                                                  GGAGTTATTTTAACCTTTTTGACAGGCGCTTTAAATATTCTTTTTTTG12715                         GlyValIleLeuThrPheLeuThrGlyAlaLeuAsnIleLeuPheLeu                              150155160                                                                     GTATATTTACCGCTTGGGATTACGGGCTATTTAATGTCCCTGGTTTTA12763                         ValTyrLeuProLeuGlyIleThrGlyTyrLeuMetSerLeuValLeu                              165170175                                                                     GCGAATGTAGGTACGATTCTATTTTTTGCTGGCACACTTTCCATTTGG12811                         AlaAsnValGlyThrIleLeuPhePheAlaGlyThrLeuSerIleTrp                              180185190                                                                     AAGGAAATTAGTTTTAAAATAATTGATAAAAAACTGATTTGGCAAATG12859                         LysGluIleSerPheLysIleIleAspLysLysLeuIleTrpGlnMet                              195200205                                                                     CTCTATTATGCCTTACCTTTGATTCCTAGTTCCATCCTGTGGTGGTTA12907                         LeuTyrTyrAlaLeuProLeuIleProSerSerIleLeuTrpTrpLeu                              210215220225                                                                  CTGAATGCTTCTAGTCGCTATTTCGTTTTATTCTTTTTAGGAGCAGGT12955                         LeuAsnAlaSerSerArgTyrPheValLeuPhePheLeuGlyAlaGly                              230235240                                                                     GCTAATGGTCTTTTGGCGGTCGCTACCAAAATTCCAAGTATTATTTCC13003                         AlaAsnGlyLeuLeuAlaValAlaThrLysIleProSerIleIleSer                              245250255                                                                     ATTTTTAATACGATTTTTACACAGGCGTGGCAAATTTCAGCCATAGAA13051                         IlePheAsnThrIlePheThrGlnAlaTrpGlnIleSerAlaIleGlu                              260265270                                                                     GAATATGATTCTCATCAAAAATCAAAATATTATTCGGATGTTTTTCAC13099                         GluTyrAspSerHisGlnLysSerLysTyrTyrSerAspValPheHis                              275280285                                                                     TACTTAGCAACTTTTCTATTGTTAGGGACATCAGCTTTTATGATTGTG13147                         TyrLeuAlaThrPheLeuLeuLeuGlyThrSerAlaPheMetIleVal                              290295300305                                                                  CTTAAACCAATTGTCGAAAAAGTCGTTTCAAGTGACTATGCAAGTTCA13195                         LeuLysProIleValGluLysValValSerSerAspTyrAlaSerSer                              310315320                                                                     TGGCAATATGTTCCTTTCTTTATGTTGTCGATGCTATTTTCCTCGTTT13243                         TrpGlnTyrValProPhePheMetLeuSerMetLeuPheSerSerPhe                              325330335                                                                     TCTGATTTTTTTGGGACTAATTATATTGCGGCTAAACAAACAAAAGGC13291                         SerAspPhePheGlyThrAsnTyrIleAlaAlaLysGlnThrLysGly                              340345350                                                                     GTATTTATGACATCTATCTATGGTACCATTGTTTGTGTCTTACTCCAA13339                         ValPheMetThrSerIleTyrGlyThrIleValCysValLeuLeuGln                              355360365                                                                     GTGGTGCTGCTACCCATCATCGGCTTGGATGGCGCAGGTTTATCAGCC13387                         ValValLeuLeuProIleIleGlyLeuAspGlyAlaGlyLeuSerAla                              370375380385                                                                  ATGCTTGGATTTTTAACAACGTTTTTATTGCGTGTCAAAGATACGCAA13435                         MetLeuGlyPheLeuThrThrPheLeuLeuArgValLysAspThrGln                              390395400                                                                     AAATTTGTGGTGATTCAGATTAAGTGGCGGATTTTTATCAGTAATTTA13483                         LysPheValValIleGlnIleLysTrpArgIlePheIleSerAsnLeu                              405410415                                                                     TTGATCGTTTTGGCACAAATTTTATGTTTGTTTTATCTACCGAGTGAA13531                         LeuIleValLeuAlaGlnIleLeuCysLeuPheTyrLeuProSerGlu                              420425430                                                                     TTTTTGTATTTTGGTCTTGCCCTATTATTTTGTGGCATGTTAGTGGTT13579                         PheLeuTyrPheGlyLeuAlaLeuLeuPheCysGlyMetLeuValVal                              435440445                                                                     AATCAGCGTACAATTTTATACATTATCATGGCGCTAAAAATAAAAAAT13627                         AsnGlnArgThrIleLeuTyrIleIleMetAlaLeuLysIleLysAsn                              450455460465                                                                  AAGACATTTGGAATGAAATCCTCATAAAAATAGACAGGAGGTGTATCTCGAATG13681                   LysThrPheGlyMetLysSerSer                                                      470                                                                           GTATCGAGATATATCTCCTGTCTATTTTTATGATACTTTTGTGTTAGCTCAACTCAACCG13741             CCTTTTAATCTCCCAACAACAATAATACCCAATCAAACAACCCAAAAAATTCAAGATAAT13801             ATCACTAATGGCAAATGTGCCCAAATAAAAGATAAATTGAATGGTTTCAATTACTAAAAG13861             AGTGACCAAACTGACAATGACAAACTGTTTGAAATCAGTATTGATACAGTAAAGGCCACC13921             TAAAGGAATGAAGTAGATAATATTTAGCACAGCCTCTTGAATCGTTCTGGGATCCGCTTT13981             TATAAAGTCAAAAGGATTCAGTGACATCGCCTGAAAATCCGTTATTTTAGTAAAAAGTAC14041             CATGAATAACAGTAATAAATACACACTGAAAGCAAGATAGAGATAAATAACTGAAAAATA14101             TTTGAGGTGATACTGGATACCAAACAACCAGATAATCAGCGTTAATAAGAGTATTAAAGT14161             CAATGTGGTATAGTCAAAGTGGTTAATCAACTTAGCCAGGCTTTGATAGCGAGTGAGAAC14221             GGGCATAATCAGCCAAGTAATCGTCGCATAACTCAGGATAAATGTGATCAATAAACTGCT14281             GAGGTAGATCATATATTTTCGCAACTGTTTCTAACTCCTTTTCTTGATGAGATTAACCCT14341             ATTTTAACATATTTTAAAACTGTCATGTTTTTATGAATTTAAAATAAATGTTAAAGAAAA14401             TAAAAATTCACCAGTTGGTTCTGTTGCAAAGTTTTCCAAAAAATCTATTTTAGTGTAAAA14461             TTGAGAAAAAAGACAGAGAGGACAGAGTAATGAATTATTTTAAAGGCAAACAATTCAAAA14521             AAGACGTCATTATTGTCTCTGTTGGTTACTACCTGCGTTACAATCTAAGCTATCGTTAAG14581             TTCAGGAATTGTTATATGATC14602                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 484 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerSerArgThrAsnArgLysGlnLysHisThrSerAsnGlySer                              151015                                                                        TrpGlyMetValAsnValGlyLeuThrIleLeuTyrAlaIleLeuAla                              202530                                                                        LeuValLeuLeuPheThrMetPheAsnTyrAsnPheLeuSerPheArg                              354045                                                                        PheLeuAsnIleIleIleThrIleGlyLeuLeuValValLeuAlaIle                              505560                                                                        SerIlePheLeuGlnLysThrLysLysLeuProLeuValThrThrVal                              65707580                                                                      ValLeuValIlePheSerLeuValSerLeuValGlyIlePheGlyPhe                              859095                                                                        LysGlnMetIleAspIleThrAsnArgMetAsnGlnThrAlaAlaPhe                              100105110                                                                     SerGluValGluMetSerIleValValProLysGluSerAspIleLys                              115120125                                                                     AspValSerGlnLeuThrSerValGlnAlaProThrLysValAspLys                              130135140                                                                     AsnAsnIleGluIleLeuMetSerAlaLeuLysLysAspLysLysVal                              145150155160                                                                  AspValLysValAspAspValAlaSerTyrGlnGluAlaTyrAspAsn                              165170175                                                                     LeuLysSerGlyLysSerLysAlaMetValLeuSerGlySerTyrAla                              180185190                                                                     SerLeuLeuGluSerValAspSerAsnTyrAlaSerAsnLeuLysThr                              195200205                                                                     IleTyrThrTyrLysIleLysLysLysAsnSerAsnSerAlaAsnGln                              210215220                                                                     ValAspSerArgValPheAsnIleTyrIleSerGlyIleAspThrTyr                              225230235240                                                                  GlyProIleSerThrValSerArgSerAspValAsnIleIleMetThr                              245250255                                                                     ValAsnMetAsnThrHisLysIleLeuLeuThrThrThrProArgAsp                              260265270                                                                     AlaTyrValLysIleProGlyGlyGlyAlaAspGlnTyrAspLysLeu                              275280285                                                                     ThrHisAlaGlyIleTyrGlyValGluThrSerGluGlnThrLeuGlu                              290295300                                                                     AspLeuTyrGlyIleLysLeuAspTyrTyrAlaArgIleAsnPheThr                              305310315320                                                                  SerPheLeuLysLeuIleAspGlnLeuGlyGlyValThrValHisAsn                              325330335                                                                     AspGlnAlaPheThrGlnGluLysPheAspPheProValGlyAspIle                              340345350                                                                     GlnMetAsnSerGluGlnAlaLeuGlyPheValArgGluArgTyrAsn                              355360365                                                                     LeuAspGlyGlyAspAsnAspArgGlyLysAsnGlnGluLysValIle                              370375380                                                                     SerAlaIleLeuAsnLysLeuAlaSerLeuLysSerValSerAsnPhe                              385390395400                                                                  ThrSerIleValAsnAsnLeuGlnAspSerValGlnThrAsnMetSer                              405410415                                                                     LeuAsnThrIleAsnAlaLeuAlaAsnThrGlnLeuGluSerGlySer                              420425430                                                                     LysPheThrValThrSerGlnAlaValThrGlyThrGlySerThrGly                              435440445                                                                     GlnLeuIleSerTyrAlaMetProAsnSerSerLeuTyrMetMetLys                              450455460                                                                     LeuAspAsnSerSerValGluSerAlaSerGlnAlaIleLysLysLeu                              465470475480                                                                  MetGluGluLys                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 243 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ValIleAspValHisSerHisIleValPheAspValAspAspGlyPro                              151015                                                                        GluThrLeuGluGluSerLeuAspLeuIleGlyGluSerTyrAlaGln                              202530                                                                        GlyValArgLysIleValSerThrSerHisArgArgLysGlyMetPhe                              354045                                                                        GluThrProGluAspLysIlePheAlaAsnPheLysLysValLysAla                              505560                                                                        GluAlaGluAlaLeuTyrProAspLeuThrIleTyrTyrGlyGlyGlu                              65707580                                                                      LeuTyrTyrThrSerAspIleValGluLysLeuGluLysAsnLeuIle                              859095                                                                        ProArgMetHisAsnThrGlnPheAlaLeuIleGluPheSerAlaArg                              100105110                                                                     ThrSerTrpLysGluIleHisSerGlyLeuSerAsnValLeuArgAla                              115120125                                                                     GlyValThrProIleValAlaHisIleGluArgTyrAspAlaLeuGlu                              130135140                                                                     GluAsnAlaAspArgValArgGluIleIleAsnMetGlyCysTyrThr                              145150155160                                                                  GlnValAsnSerSerHisValLeuLysProLysLeuPheGlyAspLys                              165170175                                                                     AspLysValArgLysLysArgValArgPhePheLeuGluLysAsnLeu                              180185190                                                                     ValHisMetValAlaSerAspMetHisAsnLeuGlyProArgProPro                              195200205                                                                     PheMetLysAspAlaTyrGluIleValLysLysAsnTyrGlySerLys                              210215220                                                                     ArgAlaLysAsnLeuPheIleGluAsnProLysThrLeuLeuGluAsn                              225230235240                                                                  GlnTyrLeu                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 230 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAsnGlnAspAsnThrLysSerAspGluIleAspValLeuAlaLeu                              151015                                                                        LeuHisLysLeuTrpThrLysLysLeuLeuIleLeuPheThrAlaPhe                              202530                                                                        TyrPheAlaValPheSerPheLeuGlyThrTyrPhePheIleGlnPro                              354045                                                                        ThrTyrThrSerThrThrArgIleTyrValValAsnGlnAlaThrAsp                              505560                                                                        AsnLysAsnLeuSerAlaGlnAspLeuGlnAlaGlyThrTyrLeuAla                              65707580                                                                      AsnAspTyrLysGluIleIleAlaSerAsnAspValLeuSerGluVal                              859095                                                                        IleLysAspGluLysLeuAsnLeuSerGluAlaGluLeuSerLysMet                              100105110                                                                     ValSerValAsnIleProThrAspThrArgLeuIleSerIleSerVal                              115120125                                                                     AsnAlaLysThrGlyGlnAspAlaGlnThrLeuAlaAsnLysValArg                              130135140                                                                     GluValAlaSerLysLysIleLysLysValThrLysValGluAspVal                              145150155160                                                                  ThrThrLeuGluGluAlaLysLeuProGluSerProSerSerProAsn                              165170175                                                                     IleLysLeuAsnValLeuLeuGlyAlaValLeuGlyGlyPheLeuAla                              180185190                                                                     ValValGlyValLeuValArgGluIleLeuAspAspArgValArgArg                              195200205                                                                     ProGluAspValGluAspAlaLeuGlyMetThrLeuLeuGlyIleVal                              210215220                                                                     ProAspThrAspLysIle                                                            225230                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 249 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetProLeuLeuLysLeuValLysSerLysValAspPheAlaLysLys                              151015                                                                        ThrGluGluTyrTyrAsnAlaIleArgThrAsnIleGlnPheSerGly                              202530                                                                        AlaGlnMetLysValIleAlaIleSerSerValGluAlaGlyGluGly                              354045                                                                        LysSerMetIleSerValAsnLeuAlaIleSerPheAlaSerValGly                              505560                                                                        LeuArgThrLeuLeuIleAspAlaGluThrArgAsnSerValLeuSer                              65707580                                                                      GlyThrPheLysSerAsnGluProTyrLysGlyLeuSerAsnPheLeu                              859095                                                                        SerGlyAsnAlaAspLeuAsnGluThrIleCysGlnThrAspIleSer                              100105110                                                                     GlyLeuAspValIleAlaSerGlyProValProProAsnProThrSer                              115120125                                                                     LeuLeuGlnAsnAspAsnPheArgHisLeuMetGluValAlaArgSer                              130135140                                                                     CysTyrAspTyrValIleIleAspThrProProValGlyLeuValIle                              145150155160                                                                  AspAlaValIleIleAlaHisGlnAlaAspAlaSerLeuLeuValThr                              165170175                                                                     GluAlaGlyLysIleLysArgArgPheValThrLysAlaValGluGln                              180185190                                                                     LeuValGluSerGlySerGlnPheLeuGlyValValLeuAsnLysVal                              195200205                                                                     AspMetThrValAspLysTyrGlyPheTyrGlySerTyrGlySerTyr                              210215220                                                                     GlyGluTyrGlyLysLysSerAspGlnLysGluGlyHisSerArgAla                              225230235240                                                                  HisArgArgArgLysValGlyTrpAsn                                                   245                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 227 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetSerGlnAlaLysGluGluIleSerAspValMetThrTyrSerGlu                              151015                                                                        LeuThrSerHisLysProLysIleIleTyrSerLeuIleLysArgIle                              202530                                                                        GlyAspIleLeuValSerSerIleGlyLeuIleIleLeuIleProLeu                              354045                                                                        PheLeuIleValAlaLeuIleMetLysCysSerGluProThrAlaPro                              505560                                                                        IlePhePheSerHisIleArgAsnGlyLysAsnGlyLysLysPheLys                              65707580                                                                      MetTyrLysPheArgThrMetCysGlnAspAlaGluSerIleLeuMet                              859095                                                                        LysAspThrGluLeuPheAlaLysPheLysAlaAsnGlyTyrLysLeu                              100105110                                                                     GluThrHisGluAspProArgIleThrLysIleGlyGlyIleLeuArg                              115120125                                                                     LysThrSerIleAspGluLeuProGlnLeuIleAsnValPheLeuGly                              130135140                                                                     GlnMetSerLeuValGlyProArgProLeuProAspArgGluIleIle                              145150155160                                                                  GluTyrGlyAspAsnGlnGluLysPheLeuSerValLysProGlyMet                              165170175                                                                     ThrGlyTrpTrpGlnValSerGlyArgSerThrIleGlyTyrProGlu                              180185190                                                                     ArgCysHisLeuGluLeuTyrTyrValGluLysCysCysPheThrPhe                              195200205                                                                     AspValLeuIleLeuLeuLysThrIleGlyIleValLeuLysArgVal                              210215220                                                                     GlyAlaArg                                                                     225                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 319 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetAsnGluGlnValThrPheIleLeuCysAspPheLeuValArgGlu                              151015                                                                        IleLysProLysTyrAspLeuLeuAlaTyrGlnPheIleSerLysLys                              202530                                                                        IleLysGluIleLysProAspIleValHisCysHisSerSerLysAla                              354045                                                                        GlyValIleGlyArgLeuAlaAlaLysArgArgGlyValLysLysIle                              505560                                                                        PheTyrThrProHisAlaTyrSerPheLeuAlaProGluPheSerGly                              65707580                                                                      LysLysLysPheLeuPheValGlnIleGluLysPheLeuSerArgPhe                              859095                                                                        AlaThrThrLysIlePheCysValSerIleAlaGluMetGlnAlaAla                              100105110                                                                     LeuGluValAsnLeuAspLysThrAspLysPheGlnValIleTyrAsn                              115120125                                                                     GlyLeuProGluIleAspLeuProSerLysGluThrIleArgAlaGln                              130135140                                                                     LeuGlyLeuGluLysAlaAlaValValIleGlyAsnAsnAlaLysMet                              145150155160                                                                  SerGluGlnLysAsnProMetPhePheMetGluIleAlaArgLysMet                              165170175                                                                     IleArgGlnAsnAlaAsnTrpHisPheValTrpValGlyAspGlyGln                              180185190                                                                     LeuMetProLeuPheGlnSerPheIleLysGlnAsnGlyLeuGluGly                              195200205                                                                     AsnIleHisLeuLeuGlyGluArgProAspSerGluIleValValThr                              210215220                                                                     AlaTyrAspIlePheLeuThrThrSerGlnTyrGluGlyLeuProTyr                              225230235240                                                                  AlaProIleGluAlaMetArgAlaGlyValProIleLeuAlaThrLys                              245250255                                                                     ValValGlyAsnSerGluLeuValIleGluGlyLysAsnGlyTyrLeu                              260265270                                                                     IleAspLeuGluTrpSerLysSerValGluGluLysLeuTyrLysAla                              275280285                                                                     AlaLysIleAspAlaGlnMetIleLysAlaAspPheArgGlnArgPhe                              290295300                                                                     AlaIleAspGlnIleLeuLysGlnIleGluThrIleTyrLeuAla                                 305310315                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 372 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetLysLysIleSerIleLeuHisPheSerGlnValSerGlyGlyGly                              151015                                                                        ValGluLysTyrIleLysLeuPheLeuLysTyrSerAspValThrLys                              202530                                                                        PheAsnAsnTyrLeuValAlaProAsnLeuGluAsnTyrAspGluPhe                              354045                                                                        AsnGlyTyrLeuLysMetSerValAsnPheAsnMetGluGlnThrPhe                              505560                                                                        SerProLeuLysIlePheLysAsnValPhePheIleArgSerValLeu                              65707580                                                                      LysLysIleAsnProAspIleValTyrLeuHisSerThrPheAlaGly                              859095                                                                        ValValGlyArgIleAlaSerIleGlyLeuProThrLysValValTyr                              100105110                                                                     AsnProHisGlyTrpSerPheLysMetAspAsnSerTyrLeuLysLys                              115120125                                                                     LeuIlePheLysLeuIleGluPheSerLeuSerPheLeuThrAspLys                              130135140                                                                     PheIleLeuIleSerGluSerGluTyrIleLeuAlaAsnHisIleSer                              145150155160                                                                  PheAsnLysSerLysPheSerLeuIleAsnAsnGlyValGluValIle                              165170175                                                                     ThrGlyAspSerArgAsnGluIleGluGluIlePheProAsnGluAsp                              180185190                                                                     PheIleIleGlyMetValGlyArgLeuSerProProLysGluPhePhe                              195200205                                                                     PhePheIleAspPheAlaLysLysIleLeuGlnIleArgAsnAspThr                              210215220                                                                     AsnPheIleIleValGlyAspGlyGluLeuArgSerGluIleGluArg                              225230235240                                                                  MetIleLeuAspAsnGlyLeuGlyAspLysIleTyrIleThrGlyTrp                              245250255                                                                     ValAspAsnProArgAsnTyrIleGluLysPheAspGlnAlaIleLeu                              260265270                                                                     PheSerArgTrpGluGlyLeuSerLeuThrIleAlaGluTyrMetSer                              275280285                                                                     GlnLysLysThrIleLeuAlaThrAsnIleGlyGlyIleAsnAspLeu                              290295300                                                                     IleThrAspGlyGluThrGlyMetLeuIleGluValGlyAspLeuAsn                              305310315320                                                                  SerAlaValSerLysSerPheGluLeuArgAsnAsnLysGluValSer                              325330335                                                                     AsnGlnLeuAlaAsnAsnAlaTyrAsnLysValValGluGlnPheSer                              340345350                                                                     IleGluLysGlnMetAlaGluIleGluSerLeuPheIleGluMetCys                              355360365                                                                     AsnAsnGluLys                                                                  370                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 159 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetLeuIleLeuLysLeuLysPheHisLeuLysSerLeuPheLeuLys                              151015                                                                        TrpIleTyrArgLeuLeuTyrLeuLysLysPheGlnPheGlyAlaArg                              202530                                                                        LeuThrPheArgAspGlyPheHisLeuLeuIleGluLysSerGlyLys                              354045                                                                        ValIleIleGlyAsnHisValPhePheAsnAsnPheCysSerIleAsn                              505560                                                                        AlaMetLeuSerValThrIleGlyAspAspCysIlePheGlyGluAsn                              65707580                                                                      ValLysIleTyrAspHisAsnHisCysTyrGlnAsnLysSerGlnPro                              859095                                                                        IleSerLysGlnGlyPheSerThrAlaAlaIleGlnIleGlyArgAsn                              100105110                                                                     CysTrpIleGlySerGlnValThrIleLeuLysGlyValThrIleGly                              115120125                                                                     AspAsnSerIleIleGlyAlaGlyValValValTyrGlnAspValPro                              130135140                                                                     GluAsnSerIleValLeuSerAsnGlyGluIleArgLysArgGly                                 145150155                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 324 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetTyrLeuLysSerLeuIleSerIleValIleProValTyrAsnVal                              151015                                                                        GluLysTyrLeuGluLysCysLeuGlnSerValGlnAsnGlnThrTyr                              202530                                                                        AsnAsnPheGluValIleLeuValAsnAspGlySerThrAspSerSer                              354045                                                                        LeuSerIleCysGluLysPheValAsnGlnAspLysArgPheSerVal                              505560                                                                        PheSerLysGluAsnGlyGlyMetSerSerAlaArgAsnPheGlyIle                              65707580                                                                      LysLysAlaLysGlySerPheIleThrPheValAspSerAspAspTyr                              859095                                                                        IleValLysAspTyrLeuSerHisLeuValAlaGlyIleLysSerGlu                              100105110                                                                     ThrSerIleValCysSerLysPhePheLeuValAspGluLysGlySer                              115120125                                                                     LeuLeuThrLysLysGluAlaProLysLysLysSerGluValValSer                              130135140                                                                     IleGluGluSerIleLysIleLeuLeuLeuGlnGlnAsnGlyTyrAsp                              145150155160                                                                  LeuAlaValTrpGlyLysLeuTyrProValSerPhePheGluThrIle                              165170175                                                                     SerPheProGluGlyLysLeuTyrGluAspMetGlyThrThrTyrLys                              180185190                                                                     LeuLeuLysLeuAlaSerGluValValPheLeuAspAlaTyrAspTyr                              195200205                                                                     AlaTyrValGlnArgProAsnSerIleMetAsnSerSerPheAsnLeu                              210215220                                                                     LysLysLeuAspIleIleGluMetValHisGluMetGluAsnAspIle                              225230235240                                                                  LeuAlaGlnPheProAsnLeuAlaLeuTyrValLysAsnArgAlaPhe                              245250255                                                                     AlaAlaGluValLysIlePheLeuGluIleProLysGluLysGluPhe                              260265270                                                                     GluGlnAlaGlnLysGlnLeuTrpHisAspIleLysLysAsnArgLys                              275280285                                                                     AlaProPheMetThrLysGlyAlaArgLeuLysAsnArgLeuGlyAla                              290295300                                                                     SerLeuSerPheLeuGlyLysSerLeuPheLeuThrIleGlyLysGln                              305310315320                                                                  LeuValAspArg                                                                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetValIleTyrPheLeuLeuPheProMetIleAlaMetIleTyrLeu                              151015                                                                        MetThrLeuLeuLeuArgGlnLysAlaGlnIleGlnLysThrIlePhe                              202530                                                                        CysValLeuThrPheGlyThrLeuGlyPheIleSerAlaSerArgAla                              354045                                                                        SerSerValGlyThrAspValThrLeuTyrGluAsnIlePheLysSer                              505560                                                                        IleAsnTyrGlyIleSerAlaGluAsnAsnTrpGlyTyrValIleTyr                              65707580                                                                      AsnLysLeuIleGlySerValPheGlyTyrThrGlyHisGluIleThr                              859095                                                                        AlaAlaAsnSerValLeuIleThrIleLeuIleGlyIlePheIleTrp                              100105110                                                                     LysValAlaGluHisTyrPheValAlaThrPheLeuTyrIleSerLeu                              115120125                                                                     PheTyrTyrAlaThrSerPheAsnIleSerArgGlnPheIleAlaMet                              130135140                                                                     GlyLeuValLeuValAlaIleSerPheAlaLeuAspLysLysValMet                              145150155160                                                                  ProTrpPheIleLeuThrValLeuAlaThrLeuPheHisAlaThrAla                              165170175                                                                     IleValAlaPheProValTyrTrpLeuThrLysValHisTrpAspVal                              180185190                                                                     LysLysThrLeuSerIlePheProIleThrIlePheAlaSerPheIle                              195200205                                                                     PheAspAlaIleLeuAsnIlePheValArgPhePheProHisTyrGlu                              210215220                                                                     MetTyrIleThrGlyThrGlnPheAsnIleSerAspGlnGlyGlnGly                              225230235240                                                                  ArgValValLeuValLysIlePheIleLeuLeuIleLeuPheThrLeu                              245250255                                                                     PheLeuPheTyrLysLysSerTyrAlaLeuIleSerGluCysHisGln                              260265270                                                                     SerLeuIleAlaLeuThrThrValGlyLeuSerIleGlyIleValPhe                              275280285                                                                     TyrAsnAsnIleLeuLeuAsnArgIleGluMetPheTyrSerIleLeu                              290295300                                                                     SerIleValPheIleProIleAlaIleAspTyrIleSerLeuLysPhe                              305310315320                                                                  LysGlnLysAspAlaValArgLeuMetLeuThrIleGlyIleLeuLeu                              325330335                                                                     IleThrLeuValProTyrTyrIleGlnValSerGlyAsnTyrSerGly                              340345350                                                                     IleLeuProTyrValIleGlnGln                                                      355360                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 316 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetGluAspArgLysLysGlnValIleLeuIleLeuSerHisArgAsn                              151015                                                                        ThrLeuAlaLeuLysSerThrIleGluLeuLeuAspSerGlnTyrPhe                              202530                                                                        AspPhePheLeuHisIleAspLysLysSerArgIleGlnAspPhePhe                              354045                                                                        TyrLeuLysLysIleThrLysPheSerThrIleHisPheSerGluArg                              505560                                                                        LysAsnValHisTrpGlyGlyPheSerMetValGluAlaMetPheAla                              65707580                                                                      LeuLeuGluCysAlaArgAspThrGlyGluTyrSerTyrPheHisPhe                              859095                                                                        LeuSerGlyAspAspMetProIleLysAspAsnGluIleValPheAsn                              100105110                                                                     PhePheGluAsnSerTyrProLysAsnPheIleAspIleLeuAspPhe                              115120125                                                                     GluAsnValAsnLysAsnSerTyrPheTyrGluProProGluMetIle                              130135140                                                                     GluGluArgValLysTyrTyrTyrProHisMetAspIleLeuAsnArg                              145150155160                                                                  LysGlyThrAsnPheIleGlyLysLysLeuIleTyrLeuGlnLysLeu                              165170175                                                                     LeuLysValAsnArgLeuLysAsnArgGluIleGluIlePheLysGly                              180185190                                                                     HisGlnTrpCysSerLeuThrAsnGlnPheValAspIleLeuLeuAsp                              195200205                                                                     LysGluGluArgArgValGlyLysSerTyrPheSerSerSerLeuIle                              210215220                                                                     ProAspGluCysTyrPheGlnThrPheAlaMetIleLysLysValGlu                              225230235240                                                                  IleTyrGlnGlnLysAsnMetSerAlaArgLeuIleAspTrpThrArg                              245250255                                                                     GlyLysProTyrIleTrpArgGlnAspAspPhePheGluIleMetAsn                              260265270                                                                     AspLysAspSerMetPheSerArgLysPheAspGluAsnValAspArg                              275280285                                                                     LysIleIleGluGluIleTyrIleLysIleArgGlyArgSerThrAsp                              290295300                                                                     GluAlaAsnLysIleLysAspLysArgPheThrLys                                          305310315                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 473 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      MetAsnLysTyrLysLysLeuLeuSerAsnSerLeuValPheThrIle                              151015                                                                        GlyAsnLeuGlySerLysLeuLeuValPheLeuLeuValProLeuTyr                              202530                                                                        ThrTyrAlaMetThrProGlnGluTyrGlyMetAlaAspLeuTyrGln                              354045                                                                        ThrThrAlaAsnLeuLeuLeuProLeuIleThrMetAsnValPheAsp                              505560                                                                        AlaThrLeuArgPheAlaMetGluLysSerMetThrLysGluSerVal                              65707580                                                                      LeuThrAsnSerLeuValValTrpCysPheSerAlaValPheThrCys                              859095                                                                        LeuGlyAlaCysIleIleTyrAlaLeuAsnLeuSerAsnLysTrpTyr                              100105110                                                                     LeuAlaLeuLeuLeuThrPheAsnLeuPheGlnGlyGlyGlnSerIle                              115120125                                                                     LeuSerGlnTyrAlaArgGlyIleGlyLysSerLysIlePheAlaAla                              130135140                                                                     GlyGlyValIleLeuThrPheLeuThrGlyAlaLeuAsnIleLeuPhe                              145150155160                                                                  LeuValTyrLeuProLeuGlyIleThrGlyTyrLeuMetSerLeuVal                              165170175                                                                     LeuAlaAsnValGlyThrIleLeuPhePheAlaGlyThrLeuSerIle                              180185190                                                                     TrpLysGluIleSerPheLysIleIleAspLysLysLeuIleTrpGln                              195200205                                                                     MetLeuTyrTyrAlaLeuProLeuIleProSerSerIleLeuTrpTrp                              210215220                                                                     LeuLeuAsnAlaSerSerArgTyrPheValLeuPhePheLeuGlyAla                              225230235240                                                                  GlyAlaAsnGlyLeuLeuAlaValAlaThrLysIleProSerIleIle                              245250255                                                                     SerIlePheAsnThrIlePheThrGlnAlaTrpGlnIleSerAlaIle                              260265270                                                                     GluGluTyrAspSerHisGlnLysSerLysTyrTyrSerAspValPhe                              275280285                                                                     HisTyrLeuAlaThrPheLeuLeuLeuGlyThrSerAlaPheMetIle                              290295300                                                                     ValLeuLysProIleValGluLysValValSerSerAspTyrAlaSer                              305310315320                                                                  SerTrpGlnTyrValProPhePheMetLeuSerMetLeuPheSerSer                              325330335                                                                     PheSerAspPhePheGlyThrAsnTyrIleAlaAlaLysGlnThrLys                              340345350                                                                     GlyValPheMetThrSerIleTyrGlyThrIleValCysValLeuLeu                              355360365                                                                     GlnValValLeuLeuProIleIleGlyLeuAspGlyAlaGlyLeuSer                              370375380                                                                     AlaMetLeuGlyPheLeuThrThrPheLeuLeuArgValLysAspThr                              385390395400                                                                  GlnLysPheValValIleGlnIleLysTrpArgIlePheIleSerAsn                              405410415                                                                     LeuLeuIleValLeuAlaGlnIleLeuCysLeuPheTyrLeuProSer                              420425430                                                                     GluPheLeuTyrPheGlyLeuAlaLeuLeuPheCysGlyMetLeuVal                              435440445                                                                     ValAsnGlnArgThrIleLeuTyrIleIleMetAlaLeuLysIleLys                              450455460                                                                     AsnLysThrPheGlyMetLysSerSer                                                   465470                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 307 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetLysGlnIleLysSerLysIleArgAspLeuGlnAsnAsnPheThr                              151015                                                                        TyrValPheGlyLysLysThrPheLeuGlyArgGlyGluAlaIleIle                              202530                                                                        IleAspGluProGluHisGlyAsnLeuGlyAspGlnAlaIleAlaPhe                              354045                                                                        AlaGluAsnGlnPheLeuValAsnHisValSerValArgAspValGlu                              505560                                                                        HisLeuIleGluSerLysThrIleSerGluIleLysSerIleLysLys                              65707580                                                                      AsnIleGlyLysLysGluLeuValPhePheHisGlyGlyGlyAsnPhe                              859095                                                                        GlyThrLeuTyrLeuLysTyrGluArgIleArgArgLeuAlaValSer                              100105110                                                                     LysLeuProPheAsnLysMetIleLeuPheProGlnSerIleSerPhe                              115120125                                                                     GluAspSerArgPheGlyGlnLysGlnLeuAsnLysSerLysLysIle                              130135140                                                                     TyrSerGlnAsnThrAsnPheIleLeuThrAlaArgGluProLysSer                              145150155160                                                                  TyrGlyLeuMetLysLysCysPheProTyrAsnLysValIleLeuThr                              165170175                                                                     ProAspIleValLeuSerPheLysPheGluValThrIleSerAspThr                              180185190                                                                     HisIleGlyLysGluLysAspSerValIleThrTyrGluAsnArgGln                              195200205                                                                     HisTyrLeuGluIleLysTrpAspGluIleAlaGlnHisGluValAla                              210215220                                                                     LeuThrAspArgLeuHisGlyMetIlePheSerTyrIleThrGlyThr                              225230235240                                                                  ProCysValValLeuAlaAsnAsnAsnHisLysIleGluGlyThrTyr                              245250255                                                                     LysHisTrpLeuAsnGluValAsnTyrIleArgPheIleGluAsnPro                              260265270                                                                     ThrValGluAsnIleLeuAspAlaIleAsnAspLeuLysGlnIleGlu                              275280285                                                                     ProHisTyrIleAspLeuSerAspLysPheGlnProLeuIleAspAla                              290295300                                                                     IleLysGly                                                                     305                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonuceotide"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GTTGCGGCCGCGATAAAGTGTGATAAGTCCAG32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonucleotide"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ATAGCGGCCGCTTAGCTCATGTTGATGCGG30                                              (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonucleotide"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CCTGCGGCCGCGCTTCCTAATTCTGTAATCG31                                             (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonucleotide"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CTGGCGGCCGCTACTTCACGTTTCTTTGCAT31                                             (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonucleotide"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TACGCGGCCGCACATAGAATAAGGCTTTACG31                                             __________________________________________________________________________

What is claimed is:
 1. Method for the production of an exopolysaccharidecomprising cloning a DNA fragment encoding amino acid sequences of SEQID NO's:2-14 into a vector construct, transforming a bacterial host cellwith the vector construct, and culturing the transformed bacterial hostcell under suitable conditions for the production of anexopolysaccharide.
 2. Method according to claim 1, in which the vectorcomprises, in addition, a promoter sequence and a translation activatorsequence which are functional in the said host cell.
 3. Method forrestoring the production of an exopolysaccharide in vivo comprisingcloning a DNA fragment encoding at least one amino acid sequence or afragment of an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, and SEQ ID NO:14 into a vector construct, transforming abacterial host cell with the vector construct and culturing thetransformed bacterial host cell under suitable conditions for theproduction of an exopolysaccharide.
 4. Method according to claim 1,wherein the DNA fragment comprises the DNA sequence of SEQ ID NO:1. 5.Method according to claim 1, wherein the vector comprises sequencespermitting integration into the bacterial host cell.
 6. Method accordingto claim 1, wherein the bacterial host cell is a lactic bacterium. 7.Method according to claim 6, wherein the lactic bacterium is a memberselected from the group consisting of Streptococcus cremoris,Streptococcus lactis, Lactobacillus casei, subsp. casei, Lactobacillussake, Streptococcus thermophilus, Lactobacillus delbruecki subsp.bulgaricus and Lactobacillus helveticus.
 8. Method according to claim 3,wherein the DNA fragment comprises at least one gene or a fragment of atleast one gene chosen from the group of genes delimited in the nucleicacid sequence SEQ ID NO:1 by nucleotides 352-1803, 1807-2535, 2547-3239,3249-3995, 4051-4731, 4898-5854, 6425-7540, 7736-8212, 8221-9192,9285-10364, 10392-11339, 11302-12222 and 12233-13651.
 9. Methodaccording to claim 3, wherein the vector comprises a sequence permittingautonomous replication into the bacterial host cell.
 10. Methodaccording to claim 3 wherein the vector comprises a sequence permittingintegration into the bacterial host cell.
 11. Method according to claim3, wherein the bacterial host cell is a lactic bacterium.
 12. Methodaccording to claim 3, wherein the lactic bacterium is a member selectedfrom the group consisting of Streptococcus cremoris, streptococcuslactis, Lactobacillus casei, subsp. casei, Lactobacillus sake,Streptococcus thermophilus, Lactobacillus delbruecki subsp. bulgaricusand Lactobacillus helveticus.
 13. Method for the production of anexopolysaccharide comprising cloning a DNA fragment which is more than90% homologous to the nucleic acid sequence of at least one gene chosenfrom the group of genes delimited in the nucleic acid sequence of SEQ IDNO:1 by nucleotides 352-1803, 1807-2535, 2547-3239, 3249-3995,4051-4731, 4898-5854, 6425-7540, 7736-8212, 8221-9192, 9285-10364,10392-11339, 11302-12222 and 12233-13651 and which encodes a proteininvolved in the biosynthesis of an exopolysaccharide possessing therepeat structure: ##STR5## into a vector construct, transforming abacterial host cell with the vector construct, and culturing thetransformed bacterial host cell under suitable conditions for theproduction of an exopolysaccharide.
 14. Method according to claim 13,wherein the vector comprises a sequence permitting autonomousreplication in the bacterial host cell.
 15. Method according to claim13, wherein the vector comprises a sequence permitting integration intothe bacterial host cell.
 16. Method according to claim 13, wherein thebacterial host cell is a lactic bacterium.
 17. Method according to claim16, wherein the lactic bacterium is a member selected from the groupconsisting of Streptococcus cremoris, Streptococcus lactis,Lactobacillus casei, subsp. casei, Lactobacillus sake, Streptococcusthermophilus, Lactobacillus delbruecki subsp. bulgaricus andLactobacillus helveticus.
 18. Method according to claim 1, wherein thebacterial host cell does not produce an exopolysaccharide having thetetrasaccharide repeat structure: ##STR6##
 19. Method according to claim3, wherein the bacterial host cell does not produce an exopolysaccharidehaving the tetrasaccharide repeat structure: ##STR7##
 20. Methodaccording to claim 13, wherein the bacterial host cell does not producean exopolysaccharide having the tetrasaccharide repeat structure:##STR8##
 21. Method according to claim 3, in which the vector comprises,in addition, a promoter sequence and translation activator sequencewhich are functional in the bacterial host cell.